KR20150035654A - Microorganism capable of producing 1,4-BDO and method of producing 1,4-BDO using the same - Google Patents

Microorganism capable of producing 1,4-BDO and method of producing 1,4-BDO using the same Download PDF

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KR20150035654A
KR20150035654A KR20130115576A KR20130115576A KR20150035654A KR 20150035654 A KR20150035654 A KR 20150035654A KR 20130115576 A KR20130115576 A KR 20130115576A KR 20130115576 A KR20130115576 A KR 20130115576A KR 20150035654 A KR20150035654 A KR 20150035654A
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정유경
조광명
박진환
조화영
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삼성전자주식회사
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Priority to US14/500,269 priority patent/US20150093798A1/en
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Priority to US15/384,166 priority patent/US20170107544A1/en

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Abstract

Provided are a microorganism capable of producing 1,4-BDO and a method for producing 1,4-BDO using the same. According to the present invention, provided is a microorganism in which the activity of converting pyruvate to lactate, the activity of converting acetyl-CoA to ethanol, the activity of converting oxaloacetate to malate, or the combination thereof is reduced, the activity of converting succinate to 4-hydroxybutyrate is increased, and the activity of converting 4-hydroxybutyrate to 1,4-butanediol is increased.

Description

1,4-BDO 생산능을 가진 미생물 및 그를 이용한 1,4-BDO를 생산하는 방법{Microorganism capable of producing 1,4-BDO and method of producing 1,4-BDO using the same}[0001] The present invention relates to a 1,4-BDO-producing microorganism and a method for producing 1,4-BDO using the same,

1,4-BDO 생산능을 가진 미생물 및 그를 이용한 1,4-BDO를 생산하는 방법에 관한 것이다.1,4-BDO production ability and 1,4-BDO production method using the microorganism.

1,4-BDO는 용매로서 및 일부 타입의 플라스틱, 탄성 섬유 및 폴리우레탄의 제조에 산업적으로 사용될 수 있다. 유기화학에서, 1,4-BDO는 감마-부티로락톤의 합성을 위하여 사용될 수 있다. 인산 및 고온의 존재하에서, 1,4-BDO는 탈수되어 중요한 용매인 테트라히드로푸란 (THF)이 될 수 있다. 1,4-BDO can be used industrially as a solvent and in the production of some types of plastics, elastic fibers and polyurethanes. In organic chemistry, 1,4-BDO can be used for the synthesis of gamma-butyrolactone. In the presence of phosphoric acid and high temperature, 1,4-BDO can be dehydrated and become an important solvent, tetrahydrofuran (THF).

1,4-BDO는 현재 석유화학 전구체인 아세틸렌, 말레익 무수물, 및 프로필렌 옥시로부터 생산된다. 그러나, 석유 값의 상승에 따라 1,4-BDO의 대안적인 생산방법이 요구되고 있다. 1,4-BDO is currently produced from the petrochemical precursors acetylene, maleic anhydride, and propyleneoxy. However, alternative production methods of 1,4-BDO are required as the petroleum value increases.

따라서, 1,4-BDO를 미생물을 이용하여 효율적으로 제조하는 방법이 요구되고 있다. Therefore, a method for efficiently producing 1,4-BDO using microorganisms is required.

일 양상은 1,4-BDO 생산능이 향상된 미생물을 제공하는 것이다.One aspect is to provide microorganisms with enhanced 1,4-BDO production capacity.

다른 양상은 상기 미생물를 이용하여 1,4-BDO를 효율적을 생산하는 방법을 제공한다. Another aspect provides a method for efficiently producing 1,4-BDO using the microorganism.

일 양상은 피루베이트로부터 락테이트로 전환하는 활성, 아세틸 CoA를 에탄올로 전환하는 활성, 옥살로아세테이트를 말레이트로 전환하는 활성, 또는 그 조합이 감소되어 있고, 숙시네이트를 4-히드록시부티레이트로 전환하는 활성이 증가되어 있고, 4-히드록시부티레이트를 1,4-부탄디올로 전환하는 활성이 증가되어 있는 것인 미생물을 제공한다.
One aspect is that the activity of converting pyruvate to lactate, the activity of converting acetyl CoA to ethanol, the activity of converting oxaloacetate to maleate, or a combination thereof, is reduced and the succinate is reduced to 4-hydroxybutyrate Wherein the activity of converting is increased and the activity of converting 4-hydroxybutyrate to 1,4-butanediol is increased.

본 명세서에 있어서, 활성의 감소는 미생물 중의 언급된 단백질의 활성이 감소된 것일 수 있다. 상기 활성의 감소는 언급된 단백질을 코딩하는 유전자의 발현의 감소뿐만 아니라 단백질 자체의 비활성 (specific activity)의 감소와 같은 여러 원인에 의한 감소를 포함할 수 있다. 상기 활성의 감소는 언급된 단백질을 코딩하는 유전자가 불활성화되거나 감쇄에 의하여 감소되는 것일 수 있다. 상기 "감소"는 조작되지 않은 미생물에 비하여 상대적으로 감소되는 것일 수 있다. In this specification, a decrease in activity may be a decrease in the activity of the mentioned protein in the microorganism. The decrease in activity may include a decrease in the expression of the gene encoding the mentioned protein as well as a decrease in various causes such as a decrease in the specific activity of the protein itself. The decrease in activity may be that the gene encoding the mentioned protein is inactivated or attenuated. The "decrease" may be a relative decrease relative to the untreated microorganism.

본 명세서에 있어서, 용어 "불활성화 (inactivation)"는 전혀 발현이 되지 않는 유전자 또는 발현이 되더라도 그 활성이 없는 유전자가 생성되는 것을 의미할 수 있다. 용어 "감쇄 (attenuation)"는 유전자의 발현이 조작되지 않은 미생물에 비하여 낮은 수준으로 발현되거나, 또는 발현이 되더라도 그 활성이 감소되어 있는 것을 의미할 수 있다. 상기 불활성화 또는 감쇄는 상동 재조합에 의해 야기될 수 있다. 상기 불활성화 또는 감쇄는 상기 유전자의 일부 서열을 포함하는 벡터를 세포에 형질전환하고, 세포를 배양하여 상기 서열이 세포의 내인성 유전자와 상동 재조합이 일어나도록 한 후, 상동 재조합이 일어난 세포를 선발 마커에 의해 선발함으로써 이루어질 수 있다.
In the present specification, the term " inactivation "may mean that a gene is not expressed at all or that a gene having no activity is produced even if it is expressed. The term "attenuation" may mean that the expression of the gene is expressed at a lower level than that of the untreated microorganism, or that its activity is decreased even if it is expressed. The inactivation or attenuation may be caused by homologous recombination. The inactivation or attenuation may be achieved by transforming a vector containing a partial sequence of the gene into a cell, culturing the cell so that homologous recombination with the endogenous gene of the cell occurs, and then introducing the homologous recombination- As shown in FIG.

상기 미생물은, 에세리키아 (Escherichia) 속, 코리네박테리움 속, 또는 루멘 박테리아에 속하는 미생물일 수 있다. 에세리키아 속 미생물은 대장균 (E.coli)일 수 있다. 상기 미생물은 1,4-BDO 생산능을 갖는 것일 수 있다. 상기 미생물은 피루베이트로부터 락테이트로 전환하는 락테이트 데히드로게나제(LDH), 예를 들면, LDHA를 코딩하는 유전자, 아세틸 CoA를 에탄올로 전환하는 알콜 데히드로게나제 (ADH), 예를 들면, ADH1를 코딩하는 유전자, 옥살로아세테이트를 말레이트로 전환하는 말레이트 데히드로게나제를 코딩하는 유전자, 또는 그 조합은 1,4-BDO 생산하는데 충분한 정도로 불활성화 또는 감쇄되어 있다. 상기 언급된 효소들의 활성은 적절한 대조군 종, 예를 들면 조작되지 않은 미생물에 비하여, 상기 언급된 효소의 활성이 약 75%이상, 약 80%이상, 약 85%이상, 약 90%이상, 약 95%이상, 또는 약 100% 감소된 것일 수 있다. 숙시네이트를 4-히드록시부티레이트로 전환하는 활성 및/또는 4-히드록시부티레이트를 1,4-부탄디올로 전환하는 활성은 1,4-BDO 생산하는데 충분한 정도로 증가된 것일 수 있다. 상기 활성은 대조군 대비 약 110%이상, 약 120%이상, 약 130%이상, 약 140%이상, 약 150%이상, 약 160%이상, 약 170%이상, 약 200%이상, 약 300%이상, 약 500%이상, 약 1000%이상, 약 2000%이상, 또는 약 10,000%이상 증가된 것일 수 있다.
The microorganism may be a microorganism belonging to the genus Escherichia, genus Corynebacterium, or lumen bacteria. The microorganism Escherichia may be E. coli. The microorganism may be one having 1,4-BDO producing ability. The microorganism may be a lactate dehydrogenase (LDH), for example, a gene coding for LDHA, an alcohol dehydrogenase (ADH) for converting acetyl CoA to ethanol, for example, , A gene encoding ADH1, a gene encoding a malate dehydrogenase that converts oxaloacetate to maleate, or a combination thereof, is inactivated or attenuated to an extent sufficient to produce 1,4-BDO. The activity of the above-mentioned enzymes may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95% %, Or about 100%. The activity of converting succinate to 4-hydroxybutyrate and / or the activity of converting 4-hydroxybutyrate to 1,4-butanediol may be increased to an extent sufficient to produce 1,4-BDO. The activity is about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 200% About 500% or more, about 1000% or more, about 2000% or more, or about 10,000% or more.

피루베이트로부터 락테이트로 전환하는 폴리펩티드, 예를 들면, 락테이트 데히드로게나제는 NAD(P)+의 NAD(P)H로의 환원을 이용하여 피루베이트를 락테이트로 가역적으로 전환시키는 반응을 촉매하는 효소일 수 있다. 상기 락테이트 데히드로게나제는 EC.1.1.1.27 또는 EC 1.1.2.3에 속하는 효소일 수 있다. 상기 락테이트 데히드로게나제는 서열번호 1의 아미노산 서열을 가질 수 있다. 상기 락테이트 데히드로게나제를 코딩하는 유전자는 서열번호 2의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 유전자는 NADH-연결된 락테이트 데히드로게나제 (LDH)를 코딩하는 대장균 ldhA일 수 있다.
A polypeptide that converts pyruvate to lactate, for example, lactate dehydrogenase, uses a reduction of NAD (P) + to NAD (P) H to reversibly convert pyruvate to lactate, Lt; / RTI > The lactate dehydrogenase may be an enzyme belonging to EC.1.1.1.27 or EC 1.1.2.3. The lactate dehydrogenase may have the amino acid sequence of SEQ ID NO: 1. The gene encoding the lactate dehydrogenase may have the nucleotide sequence of SEQ ID NO: 2. The gene may be E. coli ldhA encoding NADH-linked lactate dehydrogenase (LDH).

아세틸 CoA를 에탄올로 전환하는 폴리펩티드는 알콜 데히드로게나제 (ADH)일 수 있다. 상기 알콜 데히드로게나제 (ADH)는 NADH의 NAD+로의 산화와 함께 아세틸 CoA를 에탄올로 가역적으로 전환하는 효소일 수 있다. 상기 알콜 데히드로게나제는 EC 1.1.1.1에 속하는 효소일 수 있다. 상기 알콜 데히드로게나제는 서열번호 3의 아미노산 서열을 가질 수 있다. 상기 알콜 데히드로게나제를 코딩하는 유전자는 서열번호 4의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 유전자는 NADH-연결된 알콜 데히드로게나제를 코딩하는 대장균 adhE일 수 있다.
The polypeptide that converts acetyl CoA to ethanol may be an alcohol dehydrogenase (ADH). The alcohol dehydrogenase (ADH) may be an enzyme that reversibly converts acetyl CoA to ethanol with the oxidation of NADH to NAD +. The alcohol dehydrogenase may be an enzyme belonging to EC 1.1.1.1. The alcohol dehydrogenase may have the amino acid sequence of SEQ ID NO: 3. The gene encoding the alcohol dehydrogenase may have the nucleotide sequence of SEQ ID NO: 4. The gene may be E. coli adhE encoding a NADH-linked alcohol dehydrogenase.

상기 말레이트 데히드로게나제는 NAD(P)+의 NAD(P)H로의 환원을 이용하여 옥살로아세테이트를 말레이트로 가역적으로 전환시키는 반응을 촉매하는 효소일 수 있다. 상기 말레이트 데히드로게나제는 EC 1.1.1.37에 속하는 것일 수 있다. 상기 말레이트 데히드로게나제는 서열번호 5의 아미노산 서열을 갖는 것일 수 있다. 상기 말레이트 데히드로게나제를 코딩하는 유전자는 서열번호 6의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 유전자는 NADH-연결된 말레이트 데히드로게나제를 코딩하는 대장균 mdh일 수 있다.
The maleate dehydrogenase may be an enzyme that catalyzes the reversible conversion of oxaloacetate to maleate using reduction of NAD (P) < + > to NAD (P) H. The maleate dehydrogenase may belong to EC 1.1.1.37. The maleate dehydrogenase may have the amino acid sequence of SEQ ID NO: 5. The gene coding for the maleate dehydrogenase may have the nucleotide sequence of SEQ ID NO: 6. The gene may be E. coli mdh encoding an NADH-linked maleate dehydrogenase.

상기 미생물에 있어서, 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드, 숙시닐 CoA를 숙시닉 세미알데히드 (SSA)로 전환하는 폴리펩티드, 숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드, 또는 그 조합의 발현의 증가에 의하여 증가된 것일 수 있다.
In the microorganism, the activity of converting the succinate to 4-hydroxybutyrate (4HB) includes a polypeptide that converts succinate to succinyl CoA, a polypeptide that converts succinyl CoA to succinic semialdehyde (SSA) May be increased by increasing the expression of a polypeptide that converts the semialdehyde (SSA) to 4-hydroxybutyrate (4HB), or a combination thereof.

상기 발현의 증가는 내재적 유전자의 발현의 증가 또는 외래 유전자의 도입에 의한 것일 수 있다. 내재적 유전자의 발현의 증가는 유전자의 증폭 또는 조절 영역의 변이에 의한 것일 수 있다. 상기 외래 유전자는 내재적 (endogenous) 또는 외재적 (exogenous) 유전자일 수 있다.
The increase in expression may be due to an increase in the expression of an endogenous gene or the introduction of a foreign gene. The increase in the expression of the endogenous gene may be due to the amplification of the gene or the variation of the regulatory region. The foreign gene may be an endogenous or exogenous gene.

숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드는 숙시네이트를 숙시닐 CoA로 가역적으로 전환하는 효소일 수 있다. 상기 효소는 숙시네이트 + CoA + NTP ↔ 숙시닐 CoA + Pi + NDP 반응을 촉매하는 것일 수 있다. NTP는 ATP 또는 GTP일 수 있다. 상기 효소는 숙시닐-CoA 신테타제 (숙시닐-CoA 리가제 또는 숙시네이트 티오키나제라고도 함)일 수 있다. 상기 효소는 숙시닐 CoA 신테타제 (SucCD) 또는 숙시닐-CoA:조효소 A 트랜스퍼라아제 (succinyl-CoA:coenzyme A transferase: Cat1)일 수 있다. 상기 숙시닐 CoA 신테타제는 EC 6.2.1 (acid-thiol ligase), 예를 들면, EC 6.2.1.4 또는 EC 6.2.1.5에 속하는 효소일 수 있다. 상기 숙시닐 CoA 신테타제 (유전자명 SucCD)는 서열번호 7의 아미노산 서열을 갖는 것일 수 있다. 상기 숙시닐 CoA 신테타제, SucCD를 코딩하는 폴리뉴클레오티드는 서열번호 8의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 Cat1은 EC. 2.8.3 (CoA-transferase), 예를 들면, EC. 2.8.3.18에 속하는 것일 수 있다. 상기 Cat1은 acetate + succinyl-CoA를 가역적으로 acetyl-CoA+ succinate로 전환하는 것일 수 있다. 상기 SucCD를 코딩하는 유전자는 대장균 유래의 것일 수 있다. 상기 Cat1은 서열번호 9의 아미노산 서열을 갖는 것일 수 있다. 상기 Cat1을 코딩하는 폴리뉴클레오티드는 서열번호 10의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 숙시닐 CoA 신테타제 (유전자명 SucCD)를 코딩하는 폴리뉴클레오티드는 대장균 유래의 것일 수 있고, 상기 Cat1을 코딩하는 유전자는 클로스트리듐 클루이베리 (Clostridium kluyveri) 유래의 것일 수 있다.
Polypeptides that convert succinate to succinyl-CoA may be enzymes that reversibly convert succinate to succinyl-CoA. The enzyme may catalyze succinate + CoA + NTP- succinyl CoA + Pi + NDP reaction. NTP can be ATP or GTP. The enzyme may be succinyl-CoA synthetase (also called succinyl-CoA ligase or succinate thiokinase). The enzyme may be succinyl-CoA synthetase (SucCD) or succinyl-CoA (coenzyme A transferase: Cat1). The succinyl CoA synthetase may be an enzyme belonging to EC 6.2.1 (acid-thiol ligase), for example, EC 6.2.1.4 or EC 6.2.1.5. The succinyl CoA synthetase (gene name SucCD) may have the amino acid sequence of SEQ ID NO: 7. The polynucleotide encoding Succinyl CoA synthetase, SucCD, may have the nucleotide sequence of SEQ ID NO: 8. Cat1 is EC. 2.8.3 (CoA-transferase), e. G., EC. 2.8.3.18. Cat1 may be reversibly converting acetate + succinyl-CoA to acetyl-CoA + succinate. The gene encoding SucCD may be derived from Escherichia coli. Cat1 may have the amino acid sequence of SEQ ID NO: 9. The polynucleotide encoding Cat1 may have the nucleotide sequence of SEQ ID NO: 10. The polynucleotide encoding the succinyl CoA synthetase (gene name SucCD) may be derived from Escherichia coli, and the gene encoding Cat1 may be derived from Clostridium kluyveri.

숙시닐 CoA를 숙시닉 세미알데히드로 전환하는 폴리펩티드는 조효소 A 의존성 숙시네이트 세미알데히드 데히드로게나제 (CoA-dependent succinate semialdehyde dehydrogenase: SucD)일 수 있다. 상기 SucD은 전자 수용체로서 NAD 또는 NADP를 사용하는 옥시도리덕타제 (EC.1.2.1), 예를 들면, 아실-CoA를 알데히드로 전환하는 효소일 수 있다. 상기 SucD는 서열번호 11의 아미노산 서열을 갖는 것일 수 있다. 상기 SucD를 코딩하는 폴리뉴클레오티드는 서열번호 12의 뉴클레오티드 서열을 가질 수 있다. 상기 숙시네이트 세미알데히드 데히드로게나제 및 그를 코딩하는 유전자는 포르피로모나스 긴기발리스 (Porphyromonas gingivalis) 유래의 것일 수 있다.The polypeptide that converts succinyl-CoA to succinic semialdehyde may be the coenzyme A-dependent succinate semialdehyde dehydrogenase (SucD). The SucD may be an oxidoreductase (EC.1.2.1) using NAD or NADP as an electron acceptor, for example, an enzyme that converts acyl-CoA to an aldehyde. The SucD may have the amino acid sequence of SEQ ID NO: 11. The polynucleotide encoding SucD may have the nucleotide sequence of SEQ ID NO: 12. The succinate semialdehyde dehydrogenase and the gene encoding it may be derived from Porphyromonas gingivalis.

숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드는 4-히드록시부티레이트 데히드로게나제 (4-hydroxybutyrate dehydrogenase: 4HBd)일 수 있다. 상기 4HBd는 전자 수용체로서 NAD 또는 NADP를 사용하는 옥시도리덕타제 (EC.1.1.1), 예를 들면, 케톤을 히드록실 또는 알데히드를 알콜로 전환하는 효소일 수 있다. 상기 4HBd는 서열번호 13의 아미노산 서열을 갖는 것일 수 있다. 상기 4HBd를 코딩하는 폴리뉴클레오티드는 서열번호 14의 뉴클레오티드 서열을 가질 수 있다. 상기 4-히드록시부티레이트 데히드로게나제 및 그를 코딩하는 유전자는 포르피로모나스 긴기발리스 (Porphyromonas gingivalis) 유래의 것일 수 있다.
The polypeptide that converts succinic semialdehyde (SSA) to 4-hydroxybutyrate (4HB) may be 4-hydroxybutyrate dehydrogenase (4HBd). The 4HBd may be an oxidoreductile agent (EC.1.1.1) using NAD or NADP as the electron acceptor, for example, an enzyme that converts ketone to hydroxyl or aldehyde to alcohol. The 4HBd may have the amino acid sequence of SEQ ID NO: 13. The polynucleotide encoding 4HBd may have the nucleotide sequence of SEQ ID NO: 14. The 4-hydroxybutyrate dehydrogenase and the gene encoding it may be derived from Porphyromonas gingivalis.

상기 미생물에 있어서, 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 예를 들면, sucCD, 숙시닐 CoA를 숙시닉 세미알데히드 (SSA)로 전환하는 폴리펩티드를 코딩하는 유전자 예를 들면, sucD, 숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드를 코딩하는 유전자 예를 들면, 4hbd, 또는 그 조합이 도입된 것일 수 있다.
In the microorganism, the activity of converting succinate to 4-hydroxybutyrate (4HB) is determined by the use of genes encoding polypeptides that convert succinate into succinyl CoA, such as sucCD, succinyl CoA in succinic semialdehyde (4Hbd), or a combination thereof, which encodes a gene encoding a polypeptide that converts a succinic acid to a sulfonylurea (SSA), for example, sucD, a polypeptide that converts succinic semialdehyde (SSA) to 4-hydroxybutyrate May be introduced.

상기 미생물에 있어서, 4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성은 4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드, 4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드, 또는 그 조합의 발현의 증가에 의하여 증가된 것일 수 있다. 상기 발현의 증가는 내재적 유전자의 발현의 증가 또는 외래 유전자의 도입에 의한 것일 수 있다. 내재적 유전자의 발현의 증가는 유전자의 증폭 또는 조절 영역의 변이에 의한 것일 수 있다. 상기 외래 유전자는 내재적 (endogenous) 또는 외재적 (exogenous) 유전자일 수 있다.
In the microorganism, the activity of converting 4-hydroxybutyrate (4HB) to 1,4-butanediol (1,4-BDO) was determined by reacting 4-hydroxybutyrate (4HB) with 4-hydroxybutyryl- (4HB-CoA), a polypeptide that converts 4-hydroxybutyryl-CoA (4HB-CoA) to 1,4-butanediol (1,4-BDO), or a combination thereof. Lt; / RTI > The increase in expression may be due to an increase in the expression of an endogenous gene or the introduction of a foreign gene. The increase in the expression of the endogenous gene may be due to the amplification of the gene or the variation of the regulatory region. The foreign gene may be an endogenous or exogenous gene.

4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드는 4-히드록시부티릴 조효소 A:아세틸 조효소 A 트랜스퍼라아제 (4-hydroxybutyryl CoA:acetyl-CoA transferase: Cat2)일 수 있다. 상기 Cat2는 CoA-트란스퍼라제 (EC.2.8.3)로 분류되는 효소일 수 있다. 상기 Cat2는 서열번호 15의 아미노산 서열을 갖는 것일 수 있다. 상기 Cat2를 코딩하는 폴리뉴클레오티드는 서열번호 16의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 4-히드록시부티릴 조효소 A:아세틸 조효소 A 트랜스퍼라아제 및 그를 코딩하는 유전자는 포르피로모나스 긴기발리스 (Porphyromonas gingivalis) 유래의 것일 수 있다.The polypeptide which converts 4-hydroxybutyrate (4HB) to 4-hydroxybutyryl-CoA (4HB-CoA) is 4-hydroxybutyryl coenzyme A: 4-hydroxybutyryl CoA: acetyl- CoA transferase: Cat2). The Cat2 may be an enzyme classified as CoA-transporter (EC.2.8.3). Cat2 may have the amino acid sequence of SEQ ID NO: 15. The polynucleotide encoding Cat2 may have the nucleotide sequence of SEQ ID NO: 16. The 4-hydroxybutyryl coenzyme A: acetyl coenzyme A transferase and the gene encoding it may be derived from Porphyromonas gingivalis.

4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드는 알코올 데히드로게나제 (alcohol dehydrogenase: AdhE2) 및/또는 알데히드 데히드로게나제 (Ald)일 수 있다. Adhe2와 Ald는 4HB-CoA로부터 4-히드록시부티르알데히드를 거쳐 1,4-BDO로 전환시킬 수 있다. 상기 AdhE2 및 Ald는 2단계로 아실-CoA를 알콜로 전환하는 효소 (EC.1.1.1) 또는 EC 1.2.1.3으로 분류되는 효소일 수 있다. 상기 AdhE2는 서열번호 17의 아미노산 서열을 갖는 것일 수 있다. 상기 AdhE2를 코딩하는 폴리뉴클레오티드는 서열번호 18의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 알코올 데히드로게나제 및 그를 코딩하는 유전자는 클로스트리듐 아세토부틸리쿰 (Clostridium acetobutylicum) 유래의 것일 수 있다. 상기 Ald는 서열번호 19의 아미노산 서열을 갖는 것일 수 있다. 상기 Ald를 코딩하는 폴리뉴클레오티드는 서열번호 20의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 알데히드 데히드로게나제 및 그를 코딩하는 유전자는 클로스트리듐 베이제링키 (Clostridium beijerinckii) 유래의 것일 수 있다.
Polypeptides that convert 4-hydroxybutyryl-CoA (4HB-CoA) to 1,4-butanediol (1,4-BDO) include alcohol dehydrogenase (AdhE2) and / or aldehyde dehydrogenase (Ald). Adhe2 and Ald can be converted from 4HB-CoA to 1,4-BDO via 4-hydroxybutyraldehyde. The AdhE2 and Ald may be enzymes which convert acyl-CoA to an alcohol in two steps (EC.1.1.1) or EC 1.2.1.3. The AdhE2 may have the amino acid sequence of SEQ ID NO: 17. The polynucleotide encoding AdhE2 may have the nucleotide sequence of SEQ ID NO: 18. The alcohol dehydrogenase and the gene encoding the alcohol dehydrogenase may be derived from Clostridium acetobutylicum. The Ald may have the amino acid sequence of SEQ ID NO: 19. The polynucleotide encoding Ald may have the nucleotide sequence of SEQ ID NO: 20. The aldehyde dehydrogenase and the gene encoding it may be derived from Clostridium beijerinckii.

4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성은 4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드를 코딩하는 유전자 예를 들면 cat2, 4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드를 코딩하는 유전자 예를 들면 ald, 또는 그 조합이 도입된 것일 수 있다. 상기 미생물은 α-케토글루타레이트 데카르복실라제를 코딩하는 유전자 (sucA)가 추가적으로 도입되지 않은 것일 수 있다.
The activity of converting 4-hydroxybutyrate (4HB) to 1,4-butanediol (1,4-BDO) was confirmed by converting 4-hydroxybutyrate (4HB) to 4-hydroxybutyryl-CoA (4HB-CoA) into 1,4-butanediol (1,4-BDO), for example, genes coding for polypeptides such as ald, Or a combination thereof may be introduced. The microorganism may be one in which a gene (sucA) encoding an? -Ketoglutarate decarboxylase is not additionally introduced.

상기 미생물은 피루베이트로부터 락테이트로 전환하는 락테이트 데히드로게나제를 코딩하는 유전자, 아세틸 CoA를 에탄올로 전환하는 알코올 데히드로게나제를 코딩하는 유전자, 및 옥살로아세테이트를 말레이트로 전환하는 말레이트 데히드로게나제를 코딩하는 유전자가, 또는 그 조합이 불활성화 또는 감쇄되어 있고, 숙시네이트를 4-히드록시부티레이트로 전환하는 활성이 증가되어 있고, 4-히드록시부티레이트를 1,4-부탄디올로 전환하는 활성이 증가되어 있는 것으로서, 숙시네이트를 4-히드록시부티레이트로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닐 CoA를 숙시닉 세미알데히드로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닉 세미알데히드를 4-히드록시부티레이트로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입되어 있고, 4-히드록시부티레이트를 4-히드록시부티릴-CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 4-히드록시부티릴-CoA를 1,4-부탄디올로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입되어 있는 것인 대장균일 수 있다.
The microorganism includes a gene encoding lactate dehydrogenase which converts pyruvate to lactate, a gene encoding alcohol dehydrogenase that converts acetyl CoA to ethanol, and a gene encoding oxaloacetate to convert malate The gene coding for latedehyde logenase, or a combination thereof, is inactivated or attenuated, the activity of converting succinate into 4-hydroxybutyrate is increased, and 4-hydroxybutyrate is converted into 1,4-butanediol , The activity of converting succinate to 4-hydroxybutyrate is increased by increasing the activity of a gene encoding a polypeptide that converts succinate to succinyl CoA, a polypeptide that encodes succinyl CoA to succinic semialdehyde , A gene encoding succinic semialdehyde to 4-hydroxybutyrate, Encoding a gene encoding a polypeptide that converts 4-hydroxybutyrate to 4-hydroxybutyryl-CoA, a gene encoding 4-hydroxybutyryl-CoA, A gene encoding a polypeptide to be transfected, or a combination thereof.

다른 양상은 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성이 증가되어 있고, 4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성이 증가되어 있는 것인 미생물을 숙시네이트의 존재하에서 배양하는 단계; 및 상기 배양물로부터 1,4-BDO를 회수하는 단계;를 포함하는, 1,4-BDO를 생산하는 방법을 제공한다.
Another aspect is the increased activity of converting succinate into 4-hydroxybutyrate (4HB), and the increased activity of converting 4-hydroxybutyrate (4HB) to 1,4-butanediol (1,4-BDO) Culturing the microorganism in the presence of succinate; And recovering 1,4-BDO from the culture. ≪ RTI ID = 0.0 > 1, < / RTI >

상기 방법은 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성이 증가되어 있고, 4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성이 증가되어 있는 것인 미생물을 숙시네이트의 존재하에서 배양하는 단계를 포함한다.
This method has an increased activity of converting succinate to 4-hydroxybutyrate (4HB) and has an increased activity of converting 4-hydroxybutyrate (4HB) to 1,4-butanediol (1,4-BDO) And culturing the microorganism in the presence of succinate.

상기 미생물은, 에세리키아 (Escherichia) 속, 코리네박테리움 속, 또는 루멘 박테리아에 속하는 미생물일 수 있다. 에세리키아 속 미생물은 대장균 (E.coli)일 수 있다. 상기 미생물은 1,4-BDO 생산능을 갖는 것일 수 있다. 상기 미생물은 피루베이트로부터 락테이트로 전환하는 락테이트 데히드로게나제(LDH), 예를 들면, LDHA를 코딩하는 유전자, 아세틸 CoA를 에탄올로 전환하는 알콜 데히드로게나제 (ADH), 예를 들면, ADH1를 코딩하는 유전자, 옥살로아세테이트를 말레이트로 전환하는 말레이트 데히드로게나제를 코딩하는 유전자, 또는 그 조합은 1,4-BDO 생산하는데 충분한 정도로 불활성화 또는 감쇄되어 있다. 상기 언급된 효소들의 활성은 적절한 대조군 종, 예를 들면 조작되지 않은 미생물에 비하여, 상기 언급된 효소의 활성이 약 75%이상, 약 80%이상, 약 85%이상, 약 90%이상, 약 95%이상, 또는 약 100% 감소된 것일 수 있다. 숙시네이트를 4-히드록시부티레이트로 전환하는 활성 및/또는 4-히드록시부티레이트를 1,4-부탄디올로 전환하는 활성은 1,4-BDO 생산하는데 충분한 정도로 증가된 것일 수 있다. 상기 활성은 대조군 대비 약 110%이상, 약 120%이상, 약 130%이상, 약 140%이상, 약 150%이상, 약 160%이상, 약 170%이상, 약 200%이상, 약 300%이상, 약 500%이상, 약 1000%이상, 약 2000%이상, 또는 약 10,000%이상 증가된 것일 수 있다.
The microorganism may be a microorganism belonging to the genus Escherichia, genus Corynebacterium, or lumen bacteria. The microorganism Escherichia may be E. coli. The microorganism may be one having 1,4-BDO producing ability. The microorganism may be a lactate dehydrogenase (LDH), for example, a gene coding for LDHA, an alcohol dehydrogenase (ADH) for converting acetyl CoA to ethanol, for example, , A gene encoding ADH1, a gene encoding a malate dehydrogenase that converts oxaloacetate to maleate, or a combination thereof, is inactivated or attenuated to an extent sufficient to produce 1,4-BDO. The activity of the above-mentioned enzymes may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95% %, Or about 100%. The activity of converting succinate to 4-hydroxybutyrate and / or the activity of converting 4-hydroxybutyrate to 1,4-butanediol may be increased to an extent sufficient to produce 1,4-BDO. The activity is about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 200% About 500% or more, about 1000% or more, about 2000% or more, or about 10,000% or more.

피루베이트로부터 락테이트로 전환하는 폴리펩티드, 예를 들면, 락테이트 데히드로게나제는 NAD(P)+의 NAD(P)H로의 환원을 이용하여 피루베이트를 락테이트로 가역적으로 전환시키는 반응을 촉매하는 효소일 수 있다. 상기 락테이트 데히드로게나제는 EC.1.1.1.27 또는 EC 1.1.2.3에 속하는 효소일 수 있다. 상기 락테이트 데히드로게나제는 서열번호 1의 아미노산 서열을 가질 수 있다. 상기 락테이트 데히드로게나제를 코딩하는 유전자는 서열번호 2의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 유전자는 NADH-연결된 락테이트 데히드로게나제 (LDH)를 코딩하는 대장균 ldhA일 수 있다.
A polypeptide that converts pyruvate to lactate, for example, lactate dehydrogenase, uses a reduction of NAD (P) + to NAD (P) H to reversibly convert pyruvate to lactate, Lt; / RTI > The lactate dehydrogenase may be an enzyme belonging to EC.1.1.1.27 or EC 1.1.2.3. The lactate dehydrogenase may have the amino acid sequence of SEQ ID NO: 1. The gene encoding the lactate dehydrogenase may have the nucleotide sequence of SEQ ID NO: 2. The gene may be E. coli ldhA encoding NADH-linked lactate dehydrogenase (LDH).

아세틸 CoA를 에탄올로 전환하는 폴리펩티드는 알콜 데히드로게나제 (ADH)일 수 있다. 상기 알콜 데히드로게나제 (ADH)는 NADH의 NAD+로의 산화와 함께 아세틸 CoA를 에탄올로 가역적으로 전환하는 효소일 수 있다. 상기 알콜 데히드로게나제는 EC 1.1.1.1에 속하는 효소일 수 있다. 상기 알콜 데히드로게나제는 서열번호 3의 아미노산 서열을 가질 수 있다. 상기 알콜 데히드로게나제를 코딩하는 유전자는 서열번호 4의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 유전자는 NADH-연결된 알콜 데히드로게나제를 코딩하는 대장균 adhE일 수 있다.
The polypeptide that converts acetyl CoA to ethanol may be an alcohol dehydrogenase (ADH). The alcohol dehydrogenase (ADH) may be an enzyme that reversibly converts acetyl CoA to ethanol with the oxidation of NADH to NAD +. The alcohol dehydrogenase may be an enzyme belonging to EC 1.1.1.1. The alcohol dehydrogenase may have the amino acid sequence of SEQ ID NO: 3. The gene encoding the alcohol dehydrogenase may have the nucleotide sequence of SEQ ID NO: 4. The gene may be E. coli adhE encoding a NADH-linked alcohol dehydrogenase.

상기 말레이트 데히드로게나제는 NAD(P)+의 NAD(P)H로의 환원을 이용하여 옥살로아세테이트를 말레이트로 가역적으로 전환시키는 반응을 촉매하는 효소일 수 있다. 상기 말레이트 데히드로게나제는 EC 1.1.1.37에 속하는 것일 수 있다. 상기 말레이트 데히드로게나제는 서열번호 5의 아미노산 서열을 갖는 것일 수 있다. 상기 말레이트 데히드로게나제를 코딩하는 유전자는 서열번호 6의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 유전자는 NADH-연결된 말레이트 데히드로게나제를 코딩하는 대장균 mdh일 수 있다.
The maleate dehydrogenase may be an enzyme that catalyzes the reversible conversion of oxaloacetate to maleate using reduction of NAD (P) < + > to NAD (P) H. The maleate dehydrogenase may belong to EC 1.1.1.37. The maleate dehydrogenase may have the amino acid sequence of SEQ ID NO: 5. The gene coding for the maleate dehydrogenase may have the nucleotide sequence of SEQ ID NO: 6. The gene may be E. coli mdh encoding an NADH-linked maleate dehydrogenase.

상기 미생물에 있어서, 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드, 숙시닐 CoA를 숙시닉 세미알데히드 (SSA)로 전환하는 폴리펩티드, 숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드, 또는 그 조합의 발현의 증가에 의하여 증가된 것일 수 있다.
In the microorganism, the activity of converting the succinate to 4-hydroxybutyrate (4HB) includes a polypeptide that converts succinate to succinyl CoA, a polypeptide that converts succinyl CoA to succinic semialdehyde (SSA) May be increased by increasing the expression of a polypeptide that converts the semialdehyde (SSA) to 4-hydroxybutyrate (4HB), or a combination thereof.

상기 발현의 증가는 내재적 유전자의 발현의 증가 또는 외래 유전자의 도입에 의한 것일 수 있다. 내재적 유전자의 발현의 증가는 유전자의 증폭 또는 조절 영역의 변이에 의한 것일 수 있다. 상기 외래 유전자는 내재적 (endogenous) 또는 외재적 (exogenous) 유전자일 수 있다.
The increase in expression may be due to an increase in the expression of an endogenous gene or the introduction of a foreign gene. The increase in the expression of the endogenous gene may be due to the amplification of the gene or the variation of the regulatory region. The foreign gene may be an endogenous or exogenous gene.

숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드는 숙시네이트를 숙시닐 CoA로 가역적으로 전환하는 효소일 수 있다. 상기 효소는 숙시네이트 + CoA + NTP ↔ 숙시닐 CoA + Pi + NDP 반응을 촉매하는 것일 수 있다. NTP는 ATP 또는 GTP일 수 있다. 상기 효소는 숙시닐-CoA 신테타제 (숙시닐-CoA 리가제 또는 숙시네이트 티오키나제라고도 함)일 수 있다. 상기 효소는 숙시닐 CoA 신테타제 (SucCD) 또는 숙시닐-CoA:조효소 A 트랜스퍼라아제 (succinyl-CoA:coenzyme A transferase: Cat1)일 수 있다. 상기 숙시닐 CoA 신테타제는 EC 6.2.1 (acid-thiol ligase), 예를 들면, EC 6.2.1.4 또는 EC 6.2.1.5에 속하는 효소일 수 있다. 상기 숙시닐 CoA 신테타제 (유전자명 SucCD)는 서열번호 7의 아미노산 서열을 갖는 것일 수 있다. 상기 숙시닐 CoA 신테타제 SucCD를 코딩하는 폴리뉴클레오티드는 서열번호 8의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 Cat1은 EC. 2.8.3 (CoA-transferase), 예를 들면, EC. 2.8.3.18에 속하는 것일 수 있다. 상기 Cat1은 acetate + succinyl-CoA를 가역적으로 acetyl-CoA+ succinate로 전환하는 것일 수 있다. 상기 SucCD를 코딩하는 유전자는 대장균 유래의 것일 수 있다. 상기 Cat1은 서열번호 9의 아미노산 서열을 갖는 것일 수 있다. 상기 Cat1을 코딩하는 폴리뉴클레오티드는 서열번호 10의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 숙시닐 CoA 신테타제 (유전자명 SucCD)를 코딩하는 폴리뉴클레오티드는 대장균 유래의 것일 수 있고, 상기 Cat1을 코딩하는 유전자는 클로스트리듐 클루이베리 (Clostridium kluyveri) 유래의 것일 수 있다.
Polypeptides that convert succinate to succinyl-CoA may be enzymes that reversibly convert succinate to succinyl-CoA. The enzyme may catalyze succinate + CoA + NTP- succinyl CoA + Pi + NDP reaction. NTP can be ATP or GTP. The enzyme may be succinyl-CoA synthetase (also called succinyl-CoA ligase or succinate thiokinase). The enzyme may be succinyl-CoA synthetase (SucCD) or succinyl-CoA (coenzyme A transferase: Cat1). The succinyl CoA synthetase may be an enzyme belonging to EC 6.2.1 (acid-thiol ligase), for example, EC 6.2.1.4 or EC 6.2.1.5. The succinyl CoA synthetase (gene name SucCD) may have the amino acid sequence of SEQ ID NO: 7. The polynucleotide encoding the succinyl CoA synthetase SucCD may be that having the nucleotide sequence of SEQ ID NO: 8. Cat1 is EC. 2.8.3 (CoA-transferase), e. G., EC. 2.8.3.18. Cat1 may be reversibly converting acetate + succinyl-CoA to acetyl-CoA + succinate. The gene encoding SucCD may be derived from Escherichia coli. Cat1 may have the amino acid sequence of SEQ ID NO: 9. The polynucleotide encoding Cat1 may have the nucleotide sequence of SEQ ID NO: 10. The polynucleotide encoding the succinyl CoA synthetase (gene name SucCD) may be derived from Escherichia coli, and the gene encoding Cat1 may be derived from Clostridium kluyveri.

숙시닐 CoA를 숙시닉 세미알데히드로 전환하는 폴리펩티드는 조효소 A 의존성 숙시네이트 세미알데히드 데히드로게나제 (CoA-dependent succinate semialdehyde dehydrogenase: SucD)일 수 있다. 상기 SucD은 전자 수용체로서 NAD 또는 NADP를 사용하는 옥시도리덕타제 (EC.1.2.1), 예를 들면, 아실-CoA를 알데히드로 전환하는 효소일 수 있다. 상기 SucD는 서열번호 11의 아미노산 서열을 갖는 것일 수 있다. 상기 SucD를 코딩하는 폴리뉴클레오티드는 서열번호 12의 뉴클레오티드 서열을 가질 수 있다. 상기 숙시네이트 세미알데히드 데히드로게나제 및 그를 코딩하는 유전자는 포르피로모나스 긴기발리스 (Porphyromonas gingivalis) 유래의 것일 수 있다.The polypeptide that converts succinyl-CoA to succinic semialdehyde may be the coenzyme A-dependent succinate semialdehyde dehydrogenase (SucD). The SucD may be an oxidoreductase (EC.1.2.1) using NAD or NADP as an electron acceptor, for example, an enzyme that converts acyl-CoA to an aldehyde. The SucD may have the amino acid sequence of SEQ ID NO: 11. The polynucleotide encoding SucD may have the nucleotide sequence of SEQ ID NO: 12. The succinate semialdehyde dehydrogenase and the gene encoding it may be derived from Porphyromonas gingivalis.

숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드는 4-히드록시부티레이트 데히드로게나제 (4-hydroxybutyrate dehydrogenase: 4HBd)일 수 있다. 상기 4HBd는 전자 수용체로서 NAD 또는 NADP를 사용하는 옥시도리덕타제 (EC.1.1.1), 예를 들면, 케톤을 히드록실 또는 알데히드를 알콜로 전환하는 효소일 수 있다. 상기 4HBd는 서열번호 13의 아미노산 서열을 갖는 것일 수 있다. 상기 4HBd를 코딩하는 폴리뉴클레오티드는 서열번호 14의 뉴클레오티드 서열을 가질 수 있다. 상기 4-히드록시부티레이트 데히드로게나제 및 그를 코딩하는 유전자는 포르피로모나스 긴기발리스 (Porphyromonas gingivalis) 유래의 것일 수 있다.
The polypeptide that converts succinic semialdehyde (SSA) to 4-hydroxybutyrate (4HB) may be 4-hydroxybutyrate dehydrogenase (4HBd). The 4HBd may be an oxidoreductile agent (EC.1.1.1) using NAD or NADP as the electron acceptor, for example, an enzyme that converts ketone to hydroxyl or aldehyde to alcohol. The 4HBd may have the amino acid sequence of SEQ ID NO: 13. The polynucleotide encoding 4HBd may have the nucleotide sequence of SEQ ID NO: 14. The 4-hydroxybutyrate dehydrogenase and the gene encoding it may be derived from Porphyromonas gingivalis.

상기 미생물에 있어서, 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 예를 들면, sucCD, 숙시닐 CoA를 숙시닉 세미알데히드 (SSA)로 전환하는 폴리펩티드를 코딩하는 유전자 예를 들면, sucD, 숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드를 코딩하는 유전자 예를 들면, 4hbd, 또는 그 조합이 도입된 것일 수 있다.
In the microorganism, the activity of converting succinate to 4-hydroxybutyrate (4HB) is determined by the use of genes encoding polypeptides that convert succinate into succinyl CoA, such as sucCD, succinyl CoA in succinic semialdehyde (4Hbd), or a combination thereof, which encodes a gene encoding a polypeptide that converts a succinic acid to a sulfonylurea (SSA), for example, sucD, a polypeptide that converts succinic semialdehyde (SSA) to 4-hydroxybutyrate May be introduced.

상기 미생물에 있어서, 4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성은 4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드, 4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드, 또는 그 조합의 발현의 증가에 의하여 증가된 것일 수 있다. 상기 발현의 증가는 내재적 유전자의 발현의 증가 또는 외래 유전자의 도입에 의한 것일 수 있다. 내재적 유전자의 발현의 증가는 유전자의 증폭 또는 조절 영역의 변이에 의한 것일 수 있다. 상기 외래 유전자는 내재적 (endogenous) 또는 외재적 (exogenous) 유전자일 수 있다.
In the microorganism, the activity of converting 4-hydroxybutyrate (4HB) to 1,4-butanediol (1,4-BDO) was determined by reacting 4-hydroxybutyrate (4HB) with 4-hydroxybutyryl- (4HB-CoA), a polypeptide that converts 4-hydroxybutyryl-CoA (4HB-CoA) to 1,4-butanediol (1,4-BDO), or a combination thereof. Lt; / RTI > The increase in expression may be due to an increase in the expression of an endogenous gene or the introduction of a foreign gene. The increase in the expression of the endogenous gene may be due to the amplification of the gene or the variation of the regulatory region. The foreign gene may be an endogenous or exogenous gene.

4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드는 4-히드록시부티릴 조효소 A:아세틸 조효소 A 트랜스퍼라아제 (4-hydroxybutyryl CoA:acetyl-CoA transferase: Cat2)일 수 있다. 상기 Cat2는 CoA-트란스퍼라제 (EC.2.8.3)로 분류되는 효소일 수 있다. 상기 Cat2는 서열번호 15의 아미노산 서열을 갖는 것일 수 있다. 상기 Cat2를 코딩하는 폴리뉴클레오티드는 서열번호 16의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 4-히드록시부티릴 조효소 A:아세틸 조효소 A 트랜스퍼라아제 및 그를 코딩하는 유전자는 포르피로모나스 긴기발리스 (Porphyromonas gingivalis) 유래의 것일 수 있다.The polypeptide which converts 4-hydroxybutyrate (4HB) to 4-hydroxybutyryl-CoA (4HB-CoA) is 4-hydroxybutyryl coenzyme A: 4-hydroxybutyryl CoA: acetyl- CoA transferase: Cat2). The Cat2 may be an enzyme classified as CoA-transporter (EC.2.8.3). Cat2 may have the amino acid sequence of SEQ ID NO: 15. The polynucleotide encoding Cat2 may have the nucleotide sequence of SEQ ID NO: 16. The 4-hydroxybutyryl coenzyme A: acetyl coenzyme A transferase and the gene encoding it may be derived from Porphyromonas gingivalis.

4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드는 알코올 데히드로게나제 (alcohol dehydrogenase: AdhE2) 및/또는 알데히드 데히드로게나제 (Ald)일 수 있다. Adhe2와 Ald는 4HB-CoA로부터 4-히드록시부티르알데히드를 거쳐 1,4-BDO로 전환시킬 수 있다. 상기 AdhE2 및 Ald는 2단계로 아실-CoA를 알콜로 전환하는 효소 (EC.1.1.1) 또는 EC 1.2.1.3으로 분류되는 효소일 수 있다. 상기 AdhE2는 서열번호 17의 아미노산 서열을 갖는 것일 수 있다. 상기 AdhE2를 코딩하는 폴리뉴클레오티드는 서열번호 18의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 알코올 데히드로게나제 및 그를 코딩하는 유전자는 클로스트리듐 아세토부틸리쿰 (Clostridium acetobutylicum) 유래의 것일 수 있다. 상기 Ald는 서열번호 19의 아미노산 서열을 갖는 것일 수 있다. 상기 Ald를 코딩하는 폴리뉴클레오티드는 서열번호 20의 뉴클레오티드 서열을 갖는 것일 수 있다. 상기 알데히드 데히드로게나제 및 그를 코딩하는 유전자는 클로스트리듐 베이제링키 (Clostridium beijerinckii) 유래의 것일 수 있다.
Polypeptides that convert 4-hydroxybutyryl-CoA (4HB-CoA) to 1,4-butanediol (1,4-BDO) include alcohol dehydrogenase (AdhE2) and / or aldehyde dehydrogenase (Ald). Adhe2 and Ald can be converted from 4HB-CoA to 1,4-BDO via 4-hydroxybutyraldehyde. The AdhE2 and Ald may be enzymes which convert acyl-CoA to an alcohol in two steps (EC.1.1.1) or EC 1.2.1.3. The AdhE2 may have the amino acid sequence of SEQ ID NO: 17. The polynucleotide encoding AdhE2 may have the nucleotide sequence of SEQ ID NO: 18. The alcohol dehydrogenase and the gene encoding the alcohol dehydrogenase may be derived from Clostridium acetobutylicum. The Ald may have the amino acid sequence of SEQ ID NO: 19. The polynucleotide encoding Ald may have the nucleotide sequence of SEQ ID NO: 20. The aldehyde dehydrogenase and the gene encoding it may be derived from Clostridium beijerinckii.

4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성은 4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드를 코딩하는 유전자 예를 들면 cat2, 4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드를 코딩하는 유전자 예를 들면 ald, 또는 그 조합이 도입된 것일 수 있다. 상기 미생물은 α-케토글루타레이트 데카르복실라제를 코딩하는 유전자 (sucA)가 추가적으로 도입되지 않은 것일 수 있다.
The activity of converting 4-hydroxybutyrate (4HB) to 1,4-butanediol (1,4-BDO) was confirmed by converting 4-hydroxybutyrate (4HB) to 4-hydroxybutyryl-CoA (4HB-CoA) into 1,4-butanediol (1,4-BDO), for example, genes coding for polypeptides such as ald, Or a combination thereof may be introduced. The microorganism may be one in which a gene (sucA) encoding an? -Ketoglutarate decarboxylase is not additionally introduced.

상기 미생물은 피루베이트로부터 락테이트로 전환하는 락테이트 데히드로게나제를 코딩하는 유전자, 아세틸 CoA를 에탄올로 전환하는 알코올 데히드로게나제를 코딩하는 유전자, 및 옥살로아세테이트를 말레이트로 전환하는 말레이트 데히드로게나제를 코딩하는 유전자가, 또는 그 조합이 불활성화 또는 감쇄되어 있고, 숙시네이트를 4-히드록시부티레이트로 전환하는 활성이 증가되어 있고, 4-히드록시부티레이트를 1,4-부탄디올로 전환하는 활성이 증가되어 있는 것으로서, 숙시네이트를 4-히드록시부티레이트로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닐 CoA를 숙시닉 세미알데히드로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닉 세미알데히드를 4-히드록시부티레이트로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입되어 있고, 4-히드록시부티레이트를 4-히드록시부티릴-CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 4-히드록시부티릴-CoA를 1,4-부탄디올로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입되어 있는 것인 대장균일 수 있다.
The microorganism includes a gene encoding lactate dehydrogenase which converts pyruvate to lactate, a gene encoding alcohol dehydrogenase that converts acetyl CoA to ethanol, and a gene encoding oxaloacetate to convert malate The gene coding for latedehyde logenase, or a combination thereof, is inactivated or attenuated, the activity of converting succinate into 4-hydroxybutyrate is increased, and 4-hydroxybutyrate is converted into 1,4-butanediol , The activity of converting succinate to 4-hydroxybutyrate is increased by increasing the activity of a gene encoding a polypeptide that converts succinate to succinyl CoA, a polypeptide that encodes succinyl CoA to succinic semialdehyde , A gene encoding succinic semialdehyde to 4-hydroxybutyrate, Encoding a gene encoding a polypeptide that converts 4-hydroxybutyrate to 4-hydroxybutyryl-CoA, a gene encoding 4-hydroxybutyryl-CoA, A gene encoding a polypeptide to be transfected, or a combination thereof.

상기 배양은 당업계에 알려진 적당한 배지와 배양조건에 따라 이루어질 수 있다. 통상의 기술자라면 선택되는 미생물에 따라 배지 및 배양조건을 용이하게 조정하여 사용할 수 있다. 배양 방법은 회분식, 연속식, 유가식, 또는 이들의 조합 배양을 포함할 수 있다. 상기 숙시네이트는 배양 중에서 피딩되는 것일 수 있다. 피딩되는 숙시네이트는 숙신산, 또는 그의 염일 수 있다. 상기 염은 소듐 염일 수 있다. 숙시네이트는 배양 시작시 또는 시작 후 1회 또는 2회 이상, 예를 들면, 수회에 공급할 수 있다. 공급 농도는 세포 농도, 공급시기 등에 따라 조절될 수 있으며, 일례로 0.1 ~ 수 백g/L, 예를 들면, 0.1 ~ 500g/L, 0.1 ~ 400g/L, 0.1 ~ 300g/L, 0.1 ~ 200g/L, 0.1 ~ 100g/L, 0.1 ~ 50g/L, 1.0 ~ 500g/L, 5.0 ~ 400g/L, 10 ~ 300g/L, 50 ~ 200g/L, 1~ 100g/L, 또는 5 ~ 50g/L,일 수 있다. 일례로, 상기 숙시네이트는 숙시네이트로부터 4-히드록시부티레이트 또는 1,4-BDO 생산 유전자가 충분히 발현되었을 때 공급될 수 있다.
The culture may be performed according to a suitable culture medium and culture conditions known in the art. As a conventional technician, the medium and the culture conditions can be easily adjusted according to the selected microorganism. The culture method may include batch, continuous, fed-batch, or combination culture thereof. The succinate may be fed in culture. The succinate to be fed may be succinic acid, or a salt thereof. The salt may be sodium salt. The succinate may be fed once or more than once, for example, several times, at the start of the culture or after the start of the culture. The supply concentration can be adjusted depending on the cell concentration, the supply timing, etc., and is, for example, from 0.1 to several hundred g / L, for example, from 0.1 to 500 g / L, from 0.1 to 400 g / L, from 0.1 to 300 g / 1 to 100 g / L, 0.1 to 50 g / L, 1.0 to 500 g / L, 5.0 to 400 g / L, 10 to 300 g / L, < / RTI > For example, the succinate may be supplied when the 4-hydroxybutyrate or 1,4-BDO producing gene from succinate is fully expressed.

상기 배지는 다양한 탄소원, 질소원 및 미량원소 성분을 포함할 수 있다. 상기 탄소원은, 예를 들면, 포도당, 자당, 유당, 과당, 말토오스, 전분, 셀룰로오스와 같은 탄수화물, 대두유, 해바라기유, 피마자유, 코코넛유와 같은 지방, 팔미트산, 스테아린산, 리놀레산과 같은 지방산, 글리세롤 및 에탄올과 같은 알코올, 아세트산과 같은 유기산, 또는 이들의 조합을 포함할 수 있다. 상기 배양은 글루코스를 탄소원으로 하여 수행될 수 있다. 상기 질소원은, 펩톤, 효모 추출물, 육즙, 맥아 추출물, 옥수수 침지액 (CSL), 및 대두밀과 같은 유기 질소원 및 요소, 황산암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄과 같은 무기 질소원, 또는 이들의 조합을 포함할 수 있다. 상기 배지는 인의 공급원으로서, 예를 들면, 인산이수소칼륨, 인산수소이칼륨 및 상응하는 소듐-함유 염, 황산마그네슘 또는 황산철과 같은 금속염을 포함할 수 있다. 또한, 아미노산, 비타민, 및 적절한 전구체 등이 배지에 포함될 수 있다. 상기 배지 또는 개별 성분은 배양액에 회분식 또는 연속식으로 첨가될 수 있다. The medium may comprise various carbon sources, nitrogen sources and trace element components. The carbon source may be selected from, for example, carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil, coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid, Alcohols such as glycerol and ethanol, organic acids such as acetic acid, or combinations thereof. The culture may be performed with glucose as a carbon source. The nitrogen source may be an organic nitrogen source such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat and an inorganic nitrogen source such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, And combinations of these. The medium can include, for example, metal salts such as potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salts, magnesium sulfate or iron sulfate as a source of phosphorus. In addition, amino acids, vitamins, and suitable precursors and the like may be included in the medium. The medium or the individual components may be added to the culture medium batchwise or continuously.

또한, 배양 중에 수산화암모늄, 수산화칼륨, 암모니아, 인산 및 황산과 같은 화합물을 미생물 배양액에 적절한 방식으로 첨가하여 배양액의 pH를 조정할 수 있다. 또한, 배양 중에 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다.
In addition, during culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the microorganism culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, bubble formation can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture.

상기 배양은 호기성, 미호기성, 또는 혐기성 조건에서 수행하는 것일 수 있다. 용어 "호기성 조건"은 배지가 산소 함유 공기와 교환가능한 상태에서 배양되는 것일 수 있다. 또한, 용어 "호기성 조건"은 배지 중 용존 산소 농도가 예를 들면, 포화 농도의 약 1%이상, 약 10%이상, 약 30%이상, 약 40%이상, 약 50%이상, 약 60%이상, 약 70%이상, 약 80%이상, 약 90%이상, 또는 약 100%의 농도에서 배양하는 것을 포함할 수 있다. 상기 배양은 배지 중 용존 산소 농도가 포화 농도의 약 1% 내지 약 100%, 약 1% 내지 약 50%, 약 50% 내지 약 100%, 약 60% 내지 약 100%, 약 70% 내지 약 100%, 약 80% 내지 약 100%, 약 90% 내지 약 100%, 약 95% 내지 약 100%, 또는 약 100%의 농도에서 배양하는 것을 포함할 수 있다. 상기 호기성 조건은 배양에 걸쳐서 유지되는 것일 수 있다. 상기 포화농도는 배양이 이루어지는 온도 예를 들면, 약 30℃에서의 포화 농도일 수 있다. 외부에서 공급되는 숙시네이트의 1,4-BDO로의 효율적인 전환을 위해서 배양 조건은 호기성일 수 있다.The culture may be carried out under aerobic, aerobic, or anaerobic conditions. The term "aerobic condition" may be that the medium is cultured in an exchangeable state with oxygen-containing air. The term "aerobic condition" is intended to mean that the dissolved oxygen concentration in the medium is at least about 1%, at least about 10%, at least about 30%, at least about 40%, at least about 50% , About 70% or more, about 80% or more, about 90% or more, or about 100%. The culture may be performed in such a manner that the dissolved oxygen concentration in the medium is between about 1% and about 100%, about 1% to about 50%, about 50% to about 100%, about 60% to about 100%, about 70% %, About 80% to about 100%, about 90% to about 100%, about 95% to about 100%, or about 100%. The aerobic condition may be maintained throughout the culture. The saturation concentration may be a temperature at which the cultivation is performed, for example, a saturated concentration at about 30 캜. The culture conditions may be aerobic for efficient conversion of externally supplied succinate to 1,4-BDO.

상기 배양에 있어서, 배양물의 온도는 20 내지 45℃, 예를 들면, 22 내지 42℃, 또는 25 내지 40℃일 수 있다. 배양 기간은 원하는 1,4-BDO의 생성량을 획득할때까지 지속될 수 있다. In this culture, the temperature of the culture may be 20 to 45 캜, for example, 22 to 42 캜, or 25 to 40 캜. The incubation period can be continued until the amount of 1,4-BDO desired is obtained.

일 양상에 따른 미생물에 의하면, 호기적 조건에서도 1,4-BDO 생산능을 가질 수 있다.According to one aspect of the microorganism, 1,4-BDO can be produced even under aerobic conditions.

다른 양상에 따른 1,4-BDO를 생산하는 방법에 의하면, 1,4-BDO를 효율적으로 생산할 수 있다.According to another method of producing 1,4-BDO, 1,4-BDO can be efficiently produced.

도 1은 일 구체예에 따른 1,4-BDO 생산용 미생물의 1,4-BDO 생산 경로를 나타낸 도면이다.
도 2는 pMloxC 벡터 지도를 나타낸 도면이다.
도 3은 pTac15k sucCD-sucD-4hbd 벡터를 나타낸 도면이다.
도 4는 pTrc99a ald-cat2 벡터를 나타낸 도면이다. FIG. 1 is a diagram showing the 1,4-BDO production path of a microorganism for 1,4-BDO production according to an embodiment.
2 is a diagram showing a pMloxC vector map.
Fig. 3 is a diagram showing the pTac15k sucCD-sucD-4hbd vector.
4 is a diagram showing the pTrc99a ald-cat2 vector.

이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

실시예Example 1: 1,4- 1: 1,4- 부탄디올을Butanediol 효율적으로 생성할 수 있는 미생물의 제작 Production of microorganisms that can be efficiently produced

본 실시예에서는 락테이트 데히드로게나제 유전자, 알코올 데히드로게나제 유전자 및 말레이트 데히드로게나제 유전자가 불활성화된, 1,4-BDO 생산능을 가지고 있는 미생물을 제작하였다. In this Example, a microorganism having a 1,4-BDO producing ability in which a lactate dehydrogenase gene, an alcohol dehydrogenase gene and a malate dehydrogenase gene were inactivated was prepared.

하기 실시예에서는 대장균 W (ATCC 9637)에서 혐기 조건에서 주요 부산물인 락테이트의 생성 경로에 관여하는 효소를 코딩하는 유전자인 ldhA (서열번호 1 및 2), 에탄올의 생성 경로에 관여하는 효소를 코딩하는 유전자인 adhE (서열번호 3 및 4), 및 숙시네이트의 생성 경로에 관여하는 효소를 코딩하는 유전자인 mdh (서열번호 5 및 6)을 결실시켰다. mdh 유전자의 결실은 내재적으로 생성된 숙시네이트가 아닌, 외부에서 공급해준 숙시네이트를 기질로 사용하여 1,4-BDO가 생산됨을 보이기 위해 수행하였다. 위와 같이 변이시킨 대장균을 숙네이트를 4-히드록시부티레이트로 전환하는 활성을 가진 폴리펩티드를 코딩하는 유전자와 4-히드록시부티레이트를 1,4-BDO로 전환하는 활성을 가진 폴리펩티드를 코딩하는 유전자를 포함하는 재조합 벡터로 형질전환함으로써 1,4-BDO를 효율적으로 생산하는 변이 대장균을 제조하였다.
In the following examples, ldhA (SEQ ID NOS: 1 and 2), which is a gene encoding an enzyme involved in lactate production pathway, which is a major by-product in anaerobic conditions in Escherichia coli W (ATCC 9637) AdhE (SEQ ID NOS: 3 and 4), and mdh (SEQ ID NOS: 5 and 6), which encode an enzyme involved in the pathway of succinate production, were deleted. The deletion of the mdh gene was carried out to show that 1,4-BDO was produced using the externally supplied succinate as the substrate, rather than the intrinsically generated succinate. A gene encoding a polypeptide having an activity of converting succinate into 4-hydroxybutyrate and a gene encoding a polypeptide having an activity of converting 4-hydroxybutyrate to 1,4-BDO To produce a mutant E. coli that efficiently produces 1,4-BDO.

1.1. 부산물 (1.1. By-products 락테이트Lactate , 에탄올, , ethanol, 숙시네이트Succinate ) 생성을 차단하기 위해 ) To block the creation ldhAldhA , adhE 및 , adhE and mdhmdh 유전자가 결실된 미생물의 제작 Production of microorganisms with deletion of genes

대장균 W (ATCC 9637)에서 아래 프라이머들로 일단계 유전자 불활성화 방법(Warner et al., PNAS, 6:97(12):6640-6645 (2000))을 이용하여 ldhA, adhE 및 mdh 유전자를 결실시키고, 얻어진 균주를 W△mdh△ldh△adhE로 명명하였다.Deletion of the ldhA, adhE and mdh genes using the one-step gene inactivation method (Warner et al., PNAS, 6: 97 (12): 6640-6645 (2000)) in E. coli W (ATCC 9637) , And the obtained strain was designated as W DELTA mdh DELTA ldh DELTA adhE.

ldhA 유전자의 결실을 위해 pMloxC 벡터 (lee, K.H. et al., Molecular systems biology 3, 149 (2007) (도 2)를 주형으로 하여 서열번호 21 및 22의 프라이머로 PCR을 수행하였다. 이때 얻어진 DNA 절편을 획득하여 람다-레드 리콤비나제 (λ-red recombinase)가 발현된 W 균주의 컴피턴트 세포 (electroporation-competent cell)에 일렉트로포레이션 (electroporation)하여 ldhA 유전자 결실 돌연변이 균주를 제작하였다. ldhA 유전자 결실의 확인을 위해 서열번호 23 및 24의 프라이머로 콜로니 PCR을 수행하였다.PCR was performed with the primers of SEQ ID NOS: 21 and 22 using the pMloxC vector (lee, KH et al., Molecular systems biology 3, 149 (2007) (Fig. 2) as a template for deletion of the ldhA gene. Was obtained and electroporation-competent cells of W strain expressing lambda-red recombinase were electroporated to produce a ldhA gene deletion mutant strain. The ldhA gene deletion Colony PCR was performed with the primers of SEQ ID NOS:

또한, adhE 유전자의 결실을 위해 같은 방법으로 서열번호 25 및 26의 프라이머가 순차적으로 사용되었고, 결실의 확인을 위해 서열번호 27 및 28의 프라이머가 사용되었다.In order to delete the adhE gene, the primers of SEQ ID NOs: 25 and 26 were used in sequence in the same manner, and the primers of SEQ ID NOs: 27 and 28 were used for confirming deletion.

또한, mdh 유전자의 결실을 위해 같은 방법으로 서열번호 29 및 30의 프라이머가 순차적으로 사용되었고, 결실의 확인을 위해 서열번호 31 및 32의 프라이머가 사용되었다.In order to delete the mdh gene, the primers of SEQ ID NOs: 29 and 30 were used sequentially in the same manner, and the primers of SEQ ID NOs: 31 and 32 were used for confirming deletion.

1.2. 1.2. sucCDsucCD , , sucDsucD , 4, 4 hbdhbd , , aldald 및  And cat2cat2 유전자의 도입 Introduction of genes

상기 1.1에서 얻은 대장균 W 돌연변이 균주 (W△mdh△ldh△adhE)에 sucCD (서열번호 7 및 8), sucD(서열번호 11 및 12), 4hbd(서열번호 13 및 14), cat2(서열번호 15 및 16), 및 ald (서열번호 17 및 18) 유전자를 도입하였다. sucCD는 대장균 유래의 유전자이고, sucD, 4hbd 및 cat2는 Porphyromonas gingivalis 유래의 것이고, ald는 Clostridium beijerinckii 유래의 것이다.
SucDNA (SEQ ID NOs: 7 and 8), sucD (SEQ ID NOs: 11 and 12), 4hbd (SEQ ID NOs: 13 and 14), and cat2 (SEQ ID NO: 15) were added to the Escherichia coli W mutant strain (WΔmdhΔldhΔadhE) And 16), and ald (SEQ ID NOS: 17 and 18) genes. sucCD is a gene derived from Escherichia coli, sucD, 4hbd and cat2 are derived from Porphyromonas gingivalis, and ald is derived from Clostridium beijerinckii.

대장균 MGM1655의 게놈 DNA를 주형으로 하고, 서열번호 33 및 34의 프라이머를 사용한 PCR을 통하여, sucCD 유전자를 얻었다. 다음으로, P. gingivalis 유래sucD, 4hbd, 및 cat2, 및 C.beijerinckii 유래의 ald 유전자는 유전자 합성 ((주)코스모진텍), 한국)을 통해 제작하였다. The sucCD gene was obtained by PCR using the genomic DNA of E. coli MGM1655 as a template and the primers of SEQ ID NOS: 33 and 34. Next, the ald gene derived from P. gingivalis, sucD, 4hbd, and cat2, and C.beijerinckii was prepared through gene synthesis (Kosmosoft Tech, Korea).

얻어진 sucCD 유전자를 제한효소 EcoRI 및 KpnI을 사용하여 pTac15k (Qian, Z. -G. et al., Biotechnol. Bioeng. 104(4):651-662 (2009))에 도입하여, pTac15k sucCD 벡터를 제작하였다. pTac15k sucCD 벡터를 제한효소 KpnI로 자른 후 이를 벡터 DNA 조각으로 사용하고, 서열번호 35 및 36의 프라이머로 PCR하여 얻어진 sucD 유전자 조각과 서열번호 37 및 38의 프라이머로 PCR하여 얻어진 4hbd 유전자 조각을 모두 주형으로 사용하여 서열번호 35 및 38의 프라이머로 PCR하여 얻어진 sucD 및 4hbd 유전자를 포함하는 DNA 조각을 삽입 DNA 조각으로 사용하여 InFusion Cloning Kit (Clontech Laboratories, Inc., USA)를 통해 연결함으로써 pTac15ksucCD-sucD-4hbd 벡터 (도 3)를 제작하였다.
The obtained sucCD gene was introduced into pTac15k (Qian, Z.-G. et al., Biotechnol. Bioeng. 104 (4): 651-662 (2009)) using restriction enzymes EcoRI and KpnI to prepare pTac15k sucCD vector Respectively. pTac15k sucCD vector was cut with restriction enzyme KpnI and used as a vector DNA fragment. The fragment of sucD gene obtained by PCR with the primers of SEQ ID NOs: 35 and 36 and the fragment of 4hbd gene obtained by PCR with the primers of SEQ ID NOS: , And the DNA fragment containing the sucD and 4hbd genes obtained by PCR with the primers of SEQ ID NOS: 35 and 38 was inserted into the InFusion Cloning Kit (Clontech Laboratories, Inc., USA) using the inserted DNA fragments to obtain pTac15ksucCD-sucD- 4hbd vector (Fig. 3).

또한, 얻어진 ald 유전자를 제한효소 NcoI 및 EcoRI을 사용하여 pTrc99a (AP Biotech)에 도입하여, pTrc99a ald 벡터를 제작하였다. pTrc99a ald 벡터를 제한효소 EcoRI 및 HindIII로 자른 후 cat2 유전자를 도입하여 pTrc99a ald-cat2 벡터 (도 4)를 제작하였다.
The obtained ald gene was introduced into pTrc99a (AP Biotech) using restriction enzymes NcoI and EcoRI to construct a pTrc99a ald vector. The pTrc99a ald vector was digested with restriction enzymes EcoRI and HindIII, and then the cat2 gene was introduced into pTrc99a ald-cat2 vector (FIG. 4).

대장균 W △mdh△ldh△adhE 돌연변이 균주에 pTac15k sucCD-sucD-4hbd 및pTrc99a ald-cat2 벡터를 열 충격 (heat shock) 방법 (Sambrook, J & Russell, D.W., New York: Cold Spring Harbor Laboratory Press, 2001)으로 도입함으로써, 1,4-BDO 생성능을 가진 균주를 제작하였다. 상기 형질전환 균주는 엠피실린 (ampicillin, 100㎍/mL) 및 카나마이신 (Kanamycin, 50㎍/mL)이 포함된 LB 평판 배지에서 선별하여 수득하였다.
The pTac15k sucCD-sucD-4hbd and pTrc99a ald-cat2 vectors were introduced into Escherichia coli WΔmdhΔldhΔadhE mutant strains by heat shock method (Sambrook, J & Russell, DW, New York: Cold Spring Harbor Laboratory Press, 2001 ) To prepare a strain having the ability to produce 1,4-BDO. The transformed strains were selected by selection on LB plate medium containing ampicillin (100 / / mL) and kanamycin (50 / / mL).

실시예Example 2: 상기 재조합 미생물의 배양을 통한  2: Through the cultivation of the recombinant microorganism 숙시네이트로부터From succinate 1,4- 1,4- BDOBDO 의 생산 확인Confirm production of

본 실시예에서는 실시예 1에서 제작한 1,4-부탄올 생성 유전자들을 포함하는 재조합 미생물의 배양을 통해 숙시네이트를 주요기질로 사용하여 1,4-부탄디올이 생산되는 것을 확인하였다. In this example, it was confirmed that 1,4-butanediol was produced using succinate as a main substrate through culturing recombinant microorganisms containing 1,4-butanol-producing genes prepared in Example 1.

구체적으로, 락테이트 데히드로게나제, 알코올 데히드로게나제 및 말레이트 데히드로게나제 유전자가 불활성화된 1,4-부탄디올 생산능을 가진 미생물을 숙시네이트 외부 공급 여부 및 공급량에 따른 1,4-부탄디올 생산능을 비교하였다.
Specifically, a microorganism having the ability to produce 1,4-butanediol, in which lactate dehydrogenase, alcohol dehydrogenase and malate dehydrogenase genes are inactivated, - butanediol production ability was compared.

2.1. 2.1. pTac15kpTac15k ( ( sucCDsucCD -- sucDsucD -4-4 hbdhbd ) 및 ) And pTrc99apTrc99a ( ( aldald -- cat2cat2 )가 도입된 재조합 대장균 W △) Introduced recombinant E. coli W? mdhmdh △△ ldhldh △△ adhEadhE 균주를 이용한  Strain 숙시네이트로부터From succinate 1,4- 1,4- BDOBDO 의 생산 확인Confirm production of

실시예 1에서 얻어진 형질전환 균주인 pTac15k (sucCD-sucD-4hbd) 및 pTrc99a (ald-cat2)가 도입된 W △mdh△ldh△adh 균주를 100μg/mL 엠피실린, 및 50μg/mL 카나마이신을 함유한 10mL LB 배지에 접종하여 30℃에서 12시간 동안 전 배양을 수행하였다. 그 후, 15 g/L 글루코스, 1 g/L 효모 추출물 (yeast extract), 100mM MOPS, 10mM NaHCO3, 100μg/mL 암피실린, 및 50μg/mL 카나마이신을 함유한 MR 배지 50mL을 함유한 250mL 플라스크에 상기 전 배양액 0.5mL를 접종하여 30℃에서 220rpm으로 진탕하면서 (shaking) 배양하였다. 상기 MR 배지는 증류수 1L당 6.67g KH2PO4, 4g (NH4)2HPO4, 0.8g citric acid, 0.8g MgSO4ㆍ7H2O, 5mL trace metal solution (증류수 1L당 10g FeSO4ㆍ7H2O, 1.35g CaCl2, 2.25g ZnSO4ㆍ7H2O, 0.5g MnSO4ㆍ4H2O, 1g CuSO4ㆍ5H2O, 0.106g (NH4)6Mo7O24ㆍ4H2O, 0.23g Na2B4O7ㆍ10H2O, 35% HCl 10mL 포함)의 성분이 있고 10N NaOH로 pH를 7.0으로 맞춘 것이다. 상기 플라스크는 막으로 된 벤트 캡 (vent cap)이 설치되어 공기가 교환되도록 되어 있다. 도입 유전자의 발현을 유도하기 위해 OD600이 0.5가 될 때까지 성장시키고, OD600이 0.5가 되었을 때 0.25 mM IPTG를 배지에 첨가하였다. IPTG 첨가 후 4 시간 동안 배양한 후 소듐 숙시네이트를 0g/L, 3g/L, 및 30g/L 농도로 각각 한번에 모두 공급한 후 48 시간 동안 동일 조건에서 배양하였다. 그 결과, 1,4-BDO 생산량 (mg/L) 및 숙시네이트 소모량 (g/L)은 표 1과 같다.The WΔmdhΔldhΔad strain into which the transformant strains pTac15k (sucCD-sucD-4hbd) and pTrc99a (ald-cat2) obtained in Example 1 were introduced was cultured in a medium containing 100 μg / ml ampicillin and 50 μg / ml kanamycin 10 mL of LB medium, and preincubated at 30 DEG C for 12 hours. Thereafter, a 250 mL flask containing 50 mL of MR medium containing 15 g / L glucose, 1 g / L yeast extract, 100 mM MOPS, 10 mM NaHCO 3 , 100 μg / mL ampicillin, and 50 μg / mL kanamycin 0.5 mL of the preculture was inoculated and cultured at 30 DEG C with shaking at 220 rpm. The MR medium contained 6.67 g KH 2 PO 4 , 4 g (NH 4 ) 2 HPO 4 , 0.8 g citric acid, 0.8 g MgSO 4揃 7H 2 O, 5 mL trace metal solution (per liter of distilled water, 10 g FeSO 4揃 7H 2 O, 1.35 g CaCl 2 , 2.25 g ZnSO 4揃 7H 2 O, 0.5 g MnSO 4揃 4H 2 O, 1 g CuSO 4揃 5H 2 O, 0.106 g (NH 4 ) 6 Mo 7 O 24揃 4H 2 O, 0.23 g Na 2 B 4 O 7 ⋅ 10 H 2 O, 10 mL of 35% HCl) and adjusting the pH to 7.0 with 10 N NaOH. The flask is provided with a vent cap which is made of a membrane so that air can be exchanged. To induce expression of the transgene was grown until the OD 600 become 0.5, OD of 0.25 mM IPTG was added to the medium when the 600 is 0.5. After 4 hours of IPTG addition, sodium succinate was fed at a concentration of 0 g / L, 3 g / L, and 30 g / L, respectively, and then cultured for 48 hours under the same conditions. Table 1 shows the 1,4-BDO production (mg / L) and succinate consumption (g / L).

1,4-BDO 및 숙시네이트의 분석 과정은 다음과 같다: 상기 배양액에서 1 mL를 취하여 13000 rpm에서 30분 동안 원심분리를 하였고, 상등액을 다시 한번 같은 조건으로 원심분리 후 800 μL를 0.45 μm 필터로 여과하여 샘플을 준비했다. 이 중에서 10 μL의 샘플을 UHPLC (Ultra High Performance Liquid Chromatography, Waters)를 이용하여 1,4-BDO의 양을 분석하였다. UHPLC는 Refractive index detector (RID)가 장착된 Agilent 1100 장치를 사용했다. 4 mM H2SO4 용액을 이동상으로 사용하고 BIO-RAD Aminex HPX-87H Column을 고정상으로 사용했으며, 이때의 유속은 0.7 ml/min이다. 컬럼과 검출기의 온도는 모두 50℃이다.The analysis procedure of 1,4-BDO and succinate is as follows: 1 mL of the culture was centrifuged at 13000 rpm for 30 minutes, and the supernatant was centrifuged again under the same conditions, and then 800 μL of 0.45 μm filter To prepare a sample. The amount of 1,4-BDO was analyzed by using UHPLC (Ultra High Performance Liquid Chromatography, Waters). UHPLC used an Agilent 1100 device equipped with a Refractive Index Detector (RID). A 4 mM H 2 SO 4 solution was used as the mobile phase and BIO-RAD Aminex HPX-87H column was used as a stationary phase with a flow rate of 0.7 ml / min. The temperature of both the column and the detector is 50 ° C.

숙시네이트 첨가량 (g/L)Amount of succinate added (g / L) 1,4-BDO 생산량 (mg/L)1,4-BDO yield (mg / L) 숙시네이트 소모량 (g/L)Succinate consumption (g / L) 00 00 00 33 3333 2.32.3 3030 173173 6.76.7

표 1에 나타낸 바와 같이, 본 실시예에 따른 배양방법에 의하면, 숙시네이트가 공급되지 않은 경우는 1,4-BDO가 생산되지 않고, 숙시네이트가 공급된 경우에만 1,4-BDO가 생산되었다. 또한, 숙시네이트 첨가양이 증가할수록 숙시네이트 소모량도 증가하고 1,4-BDO 생산량도 증가하였다.
As shown in Table 1, in the culture method according to the present embodiment, 1,4-BDO was produced only when succinate was not supplied, and 1,4-BDO was produced only when succinate was supplied . Also, as the amount of succinate added increased, the consumption of succinate increased and the amount of 1,4-BDO increased.

<110> Samsung Electronics Co. Ltd. <120> Microorganism capable of producing 1,4-BDO and method of producing 1,4-BDO using the same <130> PN100757 <160> 38 <170> KopatentIn 2.0 <210> 1 <211> 329 <212> PRT <213> E.coli <400> 1 Met Lys Leu Ala Val Tyr Ser Thr Lys Gln Tyr Asp Lys Lys Tyr Leu 1 5 10 15 Gln Gln Val Asn Glu Ser Phe Gly Phe Glu Leu Glu Phe Phe Asp Phe 20 25 30 Leu Leu Thr Glu Lys Thr Ala Lys Thr Ala Asn Gly Cys Glu Ala Val 35 40 45 Cys Ile Phe Val Asn Asp Asp Gly Ser Arg Pro Val Leu Glu Glu Leu 50 55 60 Lys Lys His Gly Val Lys Tyr Ile Ala Leu Arg Cys Ala Gly Phe Asn 65 70 75 80 Asn Val Asp Leu Asp Ala Ala Lys Glu Leu Gly Leu Lys Val Val Arg 85 90 95 Val Pro Ala Tyr Asp Pro Glu Ala Val Ala Glu His Ala Ile Gly Met 100 105 110 Met Met Thr Leu Asn Arg Arg Ile His Arg Ala Tyr Gln Arg Thr Arg 115 120 125 Asp Ala Asn Phe Ser Leu Glu Gly Leu Thr Gly Phe Thr Met Tyr Gly 130 135 140 Lys Thr Ala Gly Val Ile Gly Thr Gly Lys Ile Gly Val Ala Met Leu 145 150 155 160 Arg Ile Leu Lys Gly Phe Gly Met Arg Leu Leu Ala Phe Asp Pro Tyr 165 170 175 Pro Ser Ala Ala Ala Leu Glu Leu Gly Val Glu Tyr Val Asp Leu Pro 180 185 190 Thr Leu Phe Ser Glu Ser Asp Val Ile Ser Leu His Cys Pro Leu Thr 195 200 205 Pro Glu Asn Tyr His Leu Leu Asn Glu Ala Ala Phe Asp Gln Met Lys 210 215 220 Asn Gly Val Met Ile Val Asn Thr Ser Arg Gly Ala Leu Ile Asp Ser 225 230 235 240 Gln Ala Ala Ile Glu Ala Leu Lys Asn Gln Lys Ile Gly Ser Leu Gly 245 250 255 Met Asp Val Tyr Glu Asn Glu Arg Asp Leu Phe Phe Glu Asp Lys Ser 260 265 270 Asn Asp Val Ile Gln Asp Asp Val Phe Arg Arg Leu Ser Ala Cys His 275 280 285 Asn Val Leu Phe Thr Gly His Gln Ala Phe Leu Thr Ala Glu Ala Leu 290 295 300 Thr Ser Ile Ser Gln Thr Thr Leu Gln Asn Leu Ser Asn Leu Glu Lys 305 310 315 320 Gly Glu Thr Cys Pro Asn Glu Leu Val 325 <210> 2 <211> 990 <212> DNA <213> E.coli <400> 2 atgaaactcg ccgtttatag cacaaaacag tacgacaaga agtacctgca acaggtgaac 60 gagtcctttg gctttgagct ggaatttttt gactttctgc tgacggaaaa aaccgctaaa 120 actgccaatg gctgcgaagc ggtatgtatt ttcgtaaacg atgacggcag ccgcccggtg 180 ctggaagagc tgaaaaagca cggcgttaaa tatatcgccc tgcgctgtgc cggtttcaat 240 aacgtcgacc ttgacgcggc aaaagaactg gggctgaaag tagtccgtgt tccagcctat 300 gatccagagg ccgttgctga acacgccatc ggtatgatga tgacgctgaa ccgccgtatt 360 caccgcgcgt atcagcgtac ccgtgacgct aacttctctc tggaaggtct gaccggcttt 420 actatgtatg gcaaaacggc aggcgttatc ggtaccggta aaatcggtgt ggcgatgctg 480 cgcattctga aaggttttgg tatgcgtctg ctggcgttcg atccgtatcc aagtgcagcg 540 gcgctggaac tcggtgtgga gtatgtcgat ctgccaaccc tgttctctga atcagacgtt 600 atctctctgc actgcccgct gacaccggaa aactaccatc tgttgaacga agccgccttc 660 gatcagatga aaaatggcgt gatgatcgtc aataccagtc gcggtgcatt gattgattct 720 caggcagcaa ttgaagcgct gaaaaatcag aaaattggtt cgttgggtat ggacgtgtat 780 gagaacgaac gcgatctatt ctttgaagat aaatccaacg acgtaattca ggatgacgta 840 ttccgtcgcc tgtctgcctg ccacaacgtg ctatttaccg ggcaccaggc attcctgaca 900 gcagaagctc tgaccagtat ttctcagact acgctgcaaa acttaagcaa tctggaaaaa 960 ggcgaaacct gcccgaacga actggtttaa 990 <210> 3 <211> 891 <212> PRT <213> E.coli <400> 3 Met Ala Val Thr Asn Val Ala Glu Leu Asn Ala Leu Val Glu Arg Val 1 5 10 15 Lys Lys Ala Gln Arg Glu Tyr Ala Ser Phe Thr Gln Glu Gln Val Asp 20 25 30 Lys Ile Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro 35 40 45 Leu Ala Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val Glu Asp 50 55 60 Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr 65 70 75 80 Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp Asp Thr Phe Gly 85 90 95 Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys Gly Ile Val Pro 100 105 110 Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu 115 120 125 Lys Thr Arg Asn Ala Ile Ile Phe Ser Pro His Pro Arg Ala Lys Asp 130 135 140 Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala Ala Ile Ala Ala 145 150 155 160 Gly Ala Pro Lys Asp Leu Ile Gly Trp Ile Asp Gln Pro Ser Val Glu 165 170 175 Leu Ser Asn Ala Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala 180 185 190 Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro 195 200 205 Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp Glu Thr 210 215 220 Ala Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys Thr Phe 225 230 235 240 Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val Val Val Val Asp 245 250 255 Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Thr His Gly Gly Tyr 260 265 270 Leu Leu Gln Gly Lys Glu Leu Lys Ala Val Gln Asp Val Ile Leu Lys 275 280 285 Asn Gly Ala Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Tyr Lys Ile 290 295 300 Ala Glu Leu Ala Gly Phe Ser Val Pro Glu Asn Thr Lys Ile Leu Ile 305 310 315 320 Gly Glu Val Thr Val Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys 325 330 335 Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu Asp Ala 340 345 350 Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly His Thr 355 360 365 Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala Arg Val Ser Tyr 370 375 380 Phe Gly Gln Lys Met Lys Thr Ala Arg Ile Leu Ile Asn Thr Pro Ala 385 390 395 400 Ser Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser 405 410 415 Leu Thr Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn 420 425 430 Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala 435 440 445 Glu Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg 450 455 460 Gly Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly His Lys 465 470 475 480 Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn Gly Tyr Ala 485 490 495 Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly Val Glu Thr Glu Val 500 505 510 Phe Phe Glu Val Glu Ala Asp Pro Thr Leu Ser Ile Val Arg Lys Gly 515 520 525 Ala Glu Leu Ala Asn Ser Phe Lys Pro Asp Val Ile Ile Ala Leu Gly 530 535 540 Gly Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu 545 550 555 560 His Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile 565 570 575 Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Met 580 585 590 Ile Ala Val Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr Pro Phe 595 600 605 Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu Ala Asp 610 615 620 Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp Ala Asn Leu Val Met 625 630 635 640 Asp Met Pro Lys Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala Val Thr 645 650 655 His Ala Met Glu Ala Tyr Val Ser Val Leu Ala Ser Glu Phe Ser Asp 660 665 670 Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Tyr Leu Pro Ala 675 680 685 Ser Tyr His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg Val His 690 695 700 Ser Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu Gly 705 710 715 720 Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln Phe His Ile Pro 725 730 735 His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val Ile Arg Tyr Asn 740 745 750 Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe Ser Gln Tyr Asp Arg 755 760 765 Pro Gln Ala Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu Gly Leu 770 775 780 Ser Ala Pro Gly Asp Arg Thr Ala Ala Lys Ile Glu Lys Leu Leu Ala 785 790 795 800 Trp Leu Glu Thr Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile Arg 805 810 815 Glu Ala Gly Val Gln Glu Ala Asp Phe Leu Ala Asn Val Asp Lys Leu 820 825 830 Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr 835 840 845 Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr Tyr Gly 850 855 860 Arg Asp Tyr Val Glu Gly Glu Thr Ala Ala Lys Lys Glu Ala Ala Pro 865 870 875 880 Ala Lys Ala Glu Lys Lys Ala Lys Lys Ser Ala 885 890 <210> 4 <211> 2676 <212> DNA <213> E.coli <400> 4 atggctgtta ctaatgtcgc tgaacttaac gcactcgtag agcgtgtaaa aaaagcccag 60 cgtgaatatg ccagtttcac tcaagagcaa gtagacaaaa tcttccgcgc cgccgctctg 120 gctgctgcag atgctcgaat cccactcgcg aaaatggccg ttgccgaatc cggcatgggt 180 atcgtcgaag ataaagtgat caaaaaccac tttgcttctg aatatatcta caacgcctat 240 aaagatgaaa aaacctgtgg tgttctgtct gaagacgaca cttttggtac catcactatc 300 gctgaaccaa tcggtattat ttgcggtatc gttccgacca ctaacccgac ttcaactgct 360 atcttcaaat cgctgatcag tctgaagacc cgtaacgcca ttatcttctc cccgcacccg 420 cgtgcaaaag atgccaccaa caaagcggct gatatcgttc tgcaggctgc tatcgctgcc 480 ggtgctccga aagatctgat cggctggatc gatcaacctt ctgttgaact gtctaacgca 540 ctgatgcacc acccagacat caacctgatc ctcgcgactg gtggtccggg catggttaaa 600 gccgcataca gctccggtaa accagctatc ggtgtaggcg cgggcaacac tccagttgtt 660 atcgatgaaa ctgctgatat caaacgtgca gttgcatctg tactgatgtc caaaaccttc 720 gacaacggcg taatctgtgc ttctgaacag tctgttgttg ttgttgactc tgtttatgac 780 gctgtacgtg aacgttttgc aacccacggc ggctatctgt tgcagggtaa agagctgaaa 840 gctgttcagg atgttatcct gaaaaacggt gcgctgaacg cggctatcgt tggtcagcca 900 gcctataaaa ttgctgaact ggcaggcttc tctgtaccag aaaacaccaa gattctgatc 960 ggtgaagtga ccgttgttga tgaaagcgaa ccgttcgcac atgaaaaact gtccccgact 1020 ctggcaatgt accgcgctaa agatttcgaa gacgcggtag aaaaagcaga gaaactggtt 1080 gctatgggcg gtatcggtca tacctcttgc ctgtacactg accaggataa ccaaccggct 1140 cgcgtttctt acttcggtca gaaaatgaaa acggctcgta tcctgattaa caccccagcg 1200 tctcagggtg gtatcggtga cctgtataac ttcaaactcg caccttccct gactctgggt 1260 tgtggttctt ggggtggtaa ctccatctct gaaaacgttg gtccgaaaca cctgatcaac 1320 aagaaaaccg ttgctaagcg agctgaaaac atgttgtggc acaaacttcc gaaatctatc 1380 tacttccgcc gtggctccct gccaatcgcg ctggatgaag tgattactga tggccacaaa 1440 cgtgcgctca tcgtgactga ccgcttcctg ttcaacaatg gttatgctga tcagatcact 1500 tccgtactga aagcagcagg cgttgaaact gaagtcttct tcgaagtaga agcggacccg 1560 accctgagca tcgttcgtaa aggtgcagaa ctggcaaact ccttcaaacc agacgtgatt 1620 atcgcgctgg gtggtggttc cccgatggac gccgcgaaga tcatgtgggt tatgtacgaa 1680 catccggaaa ctcacttcga agagctggcg ctgcgcttta tggatatccg taaacgtatc 1740 tacaagttcc cgaaaatggg cgtgaaagcg aaaatgatcg ctgtcaccac cacttctggt 1800 acaggttctg aagtcactcc gtttgcggtt gtaactgacg acgctactgg tcagaaatat 1860 ccgctggcag actatgcgct gactccggat atggcgattg tcgacgccaa cctggttatg 1920 gacatgccga agtccctgtg tgctttcggt ggtctggacg cagtaactca cgccatggaa 1980 gcttatgttt ctgtactggc atctgagttc tctgatggtc aggctctgca ggcactgaaa 2040 ctgctgaaag aatatctgcc agcgtcctac cacgaagggt ctaaaaatcc ggtagcgcgt 2100 gaacgtgttc acagtgcagc gactatcgcg ggtatcgcgt ttgcgaacgc cttcctgggt 2160 gtatgtcact caatggcgca caaactgggt tcccagttcc atattccgca cggtctggca 2220 aacgccctgc tgatttgtaa cgttattcgc tacaatgcga acgacaaccc gaccaagcag 2280 actgcattca gccagtatga ccgtccgcag gctcgccgtc gttatgctga aattgccgac 2340 cacttgggtc tgagcgcacc gggcgaccgt actgctgcta agatcgagaa actgctggca 2400 tggctggaaa cgctgaaagc tgaactgggt attccgaaat ctatccgtga agctggcgtt 2460 caggaagcag acttcctggc gaacgtggat aaactgtctg aagatgcgtt cgatgaccag 2520 tgcaccggcg ctaacccgcg ttacccgctg atctccgagc tgaaacagat cctgctggat 2580 acctactacg gtcgtgatta tgtagaaggt gaaactgcag cgaaaaaaga agccgctccg 2640 gctaaagctg agaaaaaagc gaaaaaatcc gcttaa 2676 <210> 5 <211> 312 <212> PRT <213> E.coli <400> 5 Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly Ile Gly Gln Ala Leu 1 5 10 15 Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu 20 25 30 Tyr Asp Ile Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His 35 40 45 Ile Pro Thr Ala Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr 50 55 60 Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala 65 70 75 80 Arg Lys Pro Gly Met Asp Arg Ser Asp Leu Phe Asn Val Asn Ala Gly 85 90 95 Ile Val Lys Asn Leu Val Gln Gln Val Ser Lys Thr Cys Pro Lys Ala 100 105 110 Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala 115 120 125 Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe 130 135 140 Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu 145 150 155 160 Leu Lys Gly Lys Gln Pro Gly Glu Val Glu Val Pro Val Ile Gly Gly 165 170 175 His Ser Gly Val Thr Ile Leu Pro Leu Leu Ser Gln Val Pro Gly Val 180 185 190 Ser Phe Thr Glu Gln Glu Val Ala Asp Leu Thr Lys Arg Ile Gln Asn 195 200 205 Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr 210 215 220 Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg 225 230 235 240 Ala Leu Gln Gly Glu Gln Gly Val Val Glu Cys Ala Tyr Val Glu Gly 245 250 255 Asp Gly Gln Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys 260 265 270 Asn Gly Val Glu Glu Arg Lys Ser Ile Gly Thr Leu Ser Ala Phe Glu 275 280 285 Gln Ser Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala 290 295 300 Leu Gly Glu Glu Phe Val Asn Lys 305 310 <210> 6 <211> 939 <212> DNA <213> E.coli <400> 6 atgaaagtcg cagtcctcgg cgctgctggc ggtattggcc aggcgcttgc actactgtta 60 aaaacccaac tgccttcagg ttcagaactc tctctgtatg atatcgctcc agtgactccc 120 ggtgtggctg tcgatctgag ccatatccct actgctgtga aaatcaaagg tttttctggt 180 gaagatgcga ctccggcgct ggaaggcgca gatgtcgttc ttatctctgc aggtgtagcg 240 cgtaaaccgg gtatggatcg ttccgacctg tttaacgtta acgccggcat cgtgaaaaac 300 ctggtacagc aagtttcgaa aacctgcccg aaagcgtgca ttggtattat cactaacccg 360 gttaacacca cagttgcgat tgctgctgaa gtgctgaaaa aagccggtgt ttatgacaaa 420 aacaaactgt tcggcgttac cacgctggat atcattcgtt ccaacacctt tgttgcggaa 480 ctgaaaggca aacagccagg cgaagttgaa gtgccggtta ttggcggtca ctctggtgtt 540 accattctgc cgctgctgtc acaggttcct ggcgttagtt ttaccgagca ggaagtggct 600 gatctgacca aacgtatcca gaacgcaggt actgaagtgg ttgaagcgaa agccggtggc 660 gggtctgcaa ccctgtctat gggccaggca gctgcacgtt ttggtctgtc tctggtacgc 720 gcactgcagg gcgaacaagg cgttgtcgaa tgtgcctatg ttgaaggcga cggtcagtac 780 gcacgtttct tctctcaacc gctgctgctg ggtaaaaacg gcgtggaaga gcgtaaatct 840 atcggtaccc tgagcgcatt tgaacagagc gcactggaag gtatgctgga tacgctgaag 900 aaagatatcg ccctgggcga agagttcgtt aataagtaa 939 <210> 7 <211> 677 <212> PRT <213> E.coli <400> 7 Met Asn Leu His Glu Tyr Gln Ala Lys Gln Leu Phe Ala Arg Tyr Gly 1 5 10 15 Leu Pro Ala Pro Val Gly Tyr Ala Cys Thr Thr Pro Arg Glu Ala Glu 20 25 30 Glu Ala Ala Ser Lys Ile Gly Ala Gly Pro Trp Val Val Lys Cys Gln 35 40 45 Val His Ala Gly Gly Arg Gly Lys Ala Gly Gly Val Lys Val Val Asn 50 55 60 Ser Lys Glu Asp Ile Arg Ala Phe Ala Glu Asn Trp Leu Gly Lys Arg 65 70 75 80 Leu Val Thr Tyr Gln Thr Asp Ala Asn Gly Gln Pro Val Asn Gln Ile 85 90 95 Leu Val Glu Ala Ala Thr Asp Ile Ala Lys Glu Leu Tyr Leu Gly Ala 100 105 110 Val Val Asp Arg Ser Ser Arg Arg Val Val Phe Met Ala Ser Thr Glu 115 120 125 Gly Gly Val Glu Ile Glu Lys Val Ala Glu Glu Thr Pro His Leu Ile 130 135 140 His Lys Val Ala Leu Asp Pro Leu Thr Gly Pro Met Pro Tyr Gln Gly 145 150 155 160 Arg Glu Leu Ala Phe Lys Leu Gly Leu Glu Gly Lys Leu Val Gln Gln 165 170 175 Phe Thr Lys Ile Phe Met Gly Leu Ala Thr Ile Phe Leu Glu Arg Asp 180 185 190 Leu Ala Leu Ile Glu Ile Asn Pro Leu Val Ile Thr Lys Gln Gly Asp 195 200 205 Leu Ile Cys Leu Asp Gly Lys Leu Gly Ala Asp Gly Asn Ala Leu Phe 210 215 220 Arg Gln Pro Asp Leu Arg Glu Met Arg Asp Gln Ser Gln Glu Asp Pro 225 230 235 240 Arg Glu Ala Gln Ala Ala Gln Trp Glu Leu Asn Tyr Val Ala Leu Asp 245 250 255 Gly Asn Ile Gly Cys Met Val Asn Gly Ala Gly Leu Ala Met Gly Thr 260 265 270 Met Asp Ile Val Lys Leu His Gly Gly Glu Pro Ala Asn Phe Leu Asp 275 280 285 Val Gly Gly Gly Ala Thr Lys Glu Arg Val Thr Glu Ala Phe Lys Ile 290 295 300 Ile Leu Ser Asp Asp Lys Val Lys Ala Val Leu Val Asn Ile Phe Gly 305 310 315 320 Gly Ile Val Arg Cys Asp Leu Ile Ala Asp Gly Ile Ile Gly Ala Val 325 330 335 Ala Glu Val Gly Val Asn Val Pro Val Val Val Arg Leu Glu Gly Asn 340 345 350 Asn Ala Glu Leu Gly Ala Lys Lys Leu Ala Asp Ser Gly Leu Asn Ile 355 360 365 Ile Ala Ala Lys Gly Leu Thr Asp Ala Ala Gln Gln Val Val Ala Ala 370 375 380 Val Glu Gly Lys Met Ser Ile Leu Ile Asp Lys Asn Thr Lys Val Ile 385 390 395 400 Cys Gln Gly Phe Thr Gly Ser Gln Gly Thr Phe His Ser Glu Gln Ala 405 410 415 Ile Ala Tyr Gly Thr Lys Met Val Gly Gly Val Thr Pro Gly Lys Gly 420 425 430 Gly Thr Thr His Leu Gly Leu Pro Val Phe Asn Thr Val Arg Glu Ala 435 440 445 Val Ala Ala Thr Gly Ala Thr Ala Ser Val Ile Tyr Val Pro Ala Pro 450 455 460 Phe Cys Lys Asp Ser Ile Leu Glu Ala Ile Asp Ala Gly Ile Lys Leu 465 470 475 480 Ile Ile Thr Ile Thr Glu Gly Ile Pro Thr Leu Asp Met Leu Thr Val 485 490 495 Lys Val Lys Leu Asp Glu Ala Gly Val Arg Met Ile Gly Pro Asn Cys 500 505 510 Pro Gly Val Ile Thr Pro Gly Glu Cys Lys Ile Gly Ile Gln Pro Gly 515 520 525 His Ile His Lys Pro Gly Lys Val Gly Ile Val Ser Arg Ser Gly Thr 530 535 540 Leu Thr Tyr Glu Ala Val Lys Gln Thr Thr Asp Tyr Gly Phe Gly Gln 545 550 555 560 Ser Thr Cys Val Gly Ile Gly Gly Asp Pro Ile Pro Gly Ser Asn Phe 565 570 575 Ile Asp Ile Leu Glu Met Phe Glu Lys Asp Pro Gln Thr Glu Ala Ile 580 585 590 Val Met Ile Gly Glu Ile Gly Gly Ser Ala Glu Glu Glu Ala Ala Ala 595 600 605 Tyr Ile Lys Glu His Val Thr Lys Pro Val Val Gly Tyr Ile Ala Gly 610 615 620 Val Thr Ala Pro Lys Gly Lys Arg Met Gly His Ala Gly Ala Ile Ile 625 630 635 640 Ala Gly Gly Lys Gly Thr Ala Asp Glu Lys Phe Ala Ala Leu Glu Ala 645 650 655 Ala Gly Val Lys Thr Val Arg Ser Leu Ala Asp Ile Gly Glu Ala Leu 660 665 670 Lys Thr Val Leu Lys 675 <210> 8 <211> 2036 <212> DNA <213> E.coli <400> 8 atgaacttac atgaatatca ggcaaaacaa ctttttgccc gctatggctt accagcaccg 60 gtgggttatg cctgtactac tccgcgcgaa gcagaagaag ccgcttcaaa aatcggtgcc 120 ggtccgtggg tagtgaaatg tcaggttcac gctggtggcc gcggtaaagc gggcggtgtg 180 aaagttgtaa acagcaaaga agacatccgt gcttttgcag aaaactggct gggcaagcgt 240 ctggtaacgt atcaaacaga tgccaatggc caaccggtta accagattct ggttgaagca 300 gcgaccgata tcgctaaaga gctgtatctc ggtgccgttg ttgaccgtag ttcccgtcgt 360 gtggtcttta tggcctccac cgaaggcggc gtggaaatcg aaaaagtggc ggaagaaact 420 ccgcacctga tccataaagt tgcgcttgat ccgctgactg gcccgatgcc gtatcaggga 480 cgcgagctgg cgttcaaact gggtctggaa ggtaaactgg ttcagcagtt caccaaaatc 540 ttcatgggcc tggcgaccat tttcctggag cgcgacctgg cgttgatcga aatcaacccg 600 ctggtcatca ccaaacaggg cgatctgatt tgcctcgacg gcaaactggg cgctgacggc 660 aacgcactgt tccgccagcc tgatctgcgc gaaatgcgtg accagtcgca ggaagatccg 720 cgtgaagcac aggctgcaca gtgggaactg aactacgttg cgctggacgg taacatcggt 780 tgtatggtta acggcgcagg tctggcgatg ggtacgatgg acatcgttaa actgcacggc 840 ggcgaaccgg ctaacttcct tgacgttggc ggcggcgcaa ccaaagaacg tgtaaccgaa 900 gcgttcaaaa tcatcctctc tgacgacaaa gtgaaagccg ttctggttaa catcttcggc 960 ggtatcgttc gttgcgacct gatcgctgac ggtatcatcg gcgcggtagc agaagtgggt 1020 gttaacgtac cggtcgtggt acgtctggaa ggtaacaacg ccgaactcgg cgcgaagaaa 1080 ctggctgaca gcggcctgaa tattattgca gcaaaaggtc tgacggatgc agctcagcag 1140 gttgttgccg cagtggaggg gaaataatgt ccattttaat cgataaaaac accaaggtta 1200 tctgccaggg ctttaccggt agccagggga ctttccactc agaacaggcc attgcatacg 1260 gcactaaaat ggttggcggc gtaaccccag gtaaaggcgg caccacccac ctcggcctgc 1320 cggtgttcaa caccgtgcgt gaagccgttg ctgccactgg cgctaccgct tctgttatct 1380 acgtaccagc accgttctgc aaagactcca ttctggaagc catcgacgca ggcatcaaac 1440 tgattatcac catcactgaa ggcatcccga cgctggatat gctgaccgtg aaagtgaagc 1500 tggatgaagc aggcgttcgt atgatcggcc cgaactgccc aggcgttatc actccgggtg 1560 aatgcaaaat cggtatccag cctggtcaca ttcacaaacc gggtaaagtg ggtatcgttt 1620 cccgttccgg tacactgacc tatgaagcgg ttaaacagac cacggattac ggtttcggtc 1680 agtcgacctg tgtcggtatc ggcggtgacc cgatcccggg ctctaacttt atcgacattc 1740 tcgaaatgtt cgaaaaagat ccgcagaccg aagcgatcgt gatgatcggt gagatcggcg 1800 gtagcgctga agaagaagca gctgcgtaca tcaaagagca cgttaccaag ccagttgtgg 1860 gttacatcgc tggtgtgact gcgccgaaag gcaaacgtat gggccacgcg ggtgccatca 1920 ttgccggtgg gaaagggact gcggatgaga aattcgctgc tctggaagcc gcaggcgtga 1980 aaaccgttcg cagcctggcg gatatcggtg aagcactgaa aactgttctg aaataa 2036 <210> 9 <211> 538 <212> PRT <213> Clostridium kluyveri <400> 9 Met Ser Lys Gly Ile Lys Asn Ser Gln Leu Lys Lys Lys Asn Val Lys 1 5 10 15 Ala Ser Asn Val Ala Glu Lys Ile Glu Glu Lys Val Glu Lys Thr Asp 20 25 30 Lys Val Val Glu Lys Ala Ala Glu Val Thr Glu Lys Arg Ile Arg Asn 35 40 45 Leu Lys Leu Gln Glu Lys Val Val Thr Ala Asp Val Ala Ala Asp Met 50 55 60 Ile Glu Asn Gly Met Ile Val Ala Ile Ser Gly Phe Thr Pro Ser Gly 65 70 75 80 Tyr Pro Lys Glu Val Pro Lys Ala Leu Thr Lys Lys Val Asn Ala Leu 85 90 95 Glu Glu Glu Phe Lys Val Thr Leu Tyr Thr Gly Ser Ser Thr Gly Ala 100 105 110 Asp Ile Asp Gly Glu Trp Ala Lys Ala Gly Ile Ile Glu Arg Arg Ile 115 120 125 Pro Tyr Gln Thr Asn Ser Asp Met Arg Lys Lys Ile Asn Asp Gly Ser 130 135 140 Ile Lys Tyr Ala Asp Met His Leu Ser His Met Ala Gln Tyr Ile Asn 145 150 155 160 Tyr Ser Val Ile Pro Lys Val Asp Ile Ala Ile Ile Glu Ala Val Ala 165 170 175 Ile Thr Glu Glu Gly Asp Ile Ile Pro Ser Thr Gly Ile Gly Asn Thr 180 185 190 Ala Thr Phe Val Glu Asn Ala Asp Lys Val Ile Val Glu Ile Asn Glu 195 200 205 Ala Gln Pro Leu Glu Leu Glu Gly Met Ala Asp Ile Tyr Thr Leu Lys 210 215 220 Asn Pro Pro Arg Arg Glu Pro Ile Pro Ile Val Asn Ala Gly Asn Arg 225 230 235 240 Ile Gly Thr Thr Tyr Val Thr Cys Gly Ser Glu Lys Ile Cys Ala Ile 245 250 255 Val Met Thr Asn Thr Gln Asp Lys Thr Arg Pro Leu Thr Glu Val Ser 260 265 270 Pro Val Ser Gln Ala Ile Ser Asp Asn Leu Ile Gly Phe Leu Asn Lys 275 280 285 Glu Val Glu Glu Gly Lys Leu Pro Lys Asn Leu Leu Pro Ile Gln Ser 290 295 300 Gly Val Gly Ser Val Ala Asn Ala Val Leu Ala Gly Leu Cys Glu Ser 305 310 315 320 Asn Phe Lys Asn Leu Ser Cys Tyr Thr Glu Val Ile Gln Asp Ser Met 325 330 335 Leu Lys Leu Ile Lys Cys Gly Lys Ala Asp Val Val Ser Gly Thr Ser 340 345 350 Ile Ser Pro Ser Pro Glu Met Leu Pro Glu Phe Ile Lys Asp Ile Asn 355 360 365 Phe Phe Arg Glu Lys Ile Val Leu Arg Pro Gln Glu Ile Ser Asn Asn 370 375 380 Pro Glu Ile Ala Arg Arg Ile Gly Val Ile Ser Ile Asn Thr Ala Leu 385 390 395 400 Glu Val Asp Ile Tyr Gly Asn Val Asn Ser Thr His Val Met Gly Ser 405 410 415 Lys Met Met Asn Gly Ile Gly Gly Ser Gly Asp Phe Ala Arg Asn Ala 420 425 430 Tyr Leu Thr Ile Phe Thr Thr Glu Ser Ile Ala Lys Lys Gly Asp Ile 435 440 445 Ser Ser Ile Val Pro Met Val Ser His Val Asp His Thr Glu His Asp 450 455 460 Val Met Val Ile Val Thr Glu Gln Gly Val Ala Asp Leu Arg Gly Leu 465 470 475 480 Ser Pro Arg Glu Lys Ala Val Ala Ile Ile Glu Asn Cys Val His Pro 485 490 495 Asp Tyr Lys Asp Met Leu Met Glu Tyr Phe Glu Glu Ala Cys Lys Ser 500 505 510 Ser Gly Gly Asn Thr Pro His Asn Leu Glu Lys Ala Leu Ser Trp His 515 520 525 Thr Lys Phe Ile Lys Thr Gly Ser Met Lys 530 535 <210> 10 <211> 1617 <212> DNA <213> Clostridium kluyveri <400> 10 atgagtaaag ggattaagaa ctcgcaacta aaaaaaaaaa atgtgaaggc cagtaatgtg 60 gcagaaaaga ttgaagagaa agttgaaaaa acggataagg ttgttgaaaa agccgctgag 120 gttacagaga aacggattag aaacctgaag ctgcaggaga aagttgttac agcggatgtg 180 gcggctgata tgattgaaaa tggcatgatt gtggcaatca gcggttttac tccgtccggt 240 tatccaaagg aagtccctaa agcactgact aaaaaagtta atgccctgga ggaggagttc 300 aaggtcacct tatataccgg gtcaagcacg ggggccgaca tcgacgggga atgggcaaag 360 gcaggaatca tagaacggcg tatcccctac cagacaaatt ctgacatgcg aaaaaaaata 420 aatgacggtt ctattaagta cgctgatatg catttaagcc atatggctca atatattaat 480 tattctgtca ttcctaaagt cgatatagct ataatagaag cggtagctat tacggaagaa 540 ggggatataa ttccttcgac gggaattggc aataccgcga cttttgtgga aaacgcggac 600 aaagtgatag tggaaattaa cgaagcccaa ccgctggaat tggagggcat ggcagacata 660 tacacattaa aaaacccccc gcgtagagag ccgattccaa tagttaatgc tggcaatcgc 720 atagggacca catatgtgac ctgtggctcg gaaaaaatct gcgccatcgt catgacaaat 780 acgcaagaca aaacaagacc tcttacagag gtgtctcctg tatctcaggc catctccgac 840 aatctgatag gttttttaaa caaagaagtg gaagagggca aattacctaa aaacctgctc 900 cccatacagt caggagttgg tagtgtcgca aatgcggttt tggccggtct ttgtgaatca 960 aactttaaaa acctaagttg ttacacggag gttatccagg atagcatgct gaagcttata 1020 aaatgtggaa aagcagatgt ggtgtcaggc acctccataa gtccatcacc ggagatgctg 1080 cctgagttca tcaaggacat aaacttcttt agagaaaaga tagtattaag accacaggaa 1140 atcagcaata acccagagat agcacgcaga atcggtgtga tatccataaa caccgccttg 1200 gaagtagaca tatatggtaa tgtaaacagt acgcacgtta tgggaagcaa aatgatgaat 1260 ggcataggcg gttctggcga ctttgcccgc aatgcatatc tcactatctt cactacagag 1320 tctatcgcca aaaaaggcga tatctcaagc atagtgccta tggtatccca tgtggatcat 1380 accgaacatg atgtaatggt catcgttacc gaacagggag tagcggatct gcgcggtctt 1440 tctcctaggg aaaaggcggt ggctataatc gaaaattgcg ttcatccgga ctataaggat 1500 atgctgatgg agtattttga agaagcgtgc aaatcgtcag gtgggaacac cccacacaat 1560 cttgaaaaag ctctttcatg gcacacaaaa tttataaaaa cgggtagcat gaaataa 1617 <210> 11 <211> 451 <212> PRT <213> Porphyromonas gingivalis <400> 11 Met Glu Ile Lys Glu Met Val Ser Leu Ala Arg Lys Ala Gln Lys Glu 1 5 10 15 Tyr Gln Ala Thr His Asn Gln Glu Ala Val Asp Asn Ile Cys Arg Ala 20 25 30 Ala Ala Lys Val Ile Tyr Glu Asn Ala Ala Ile Leu Ala Arg Glu Ala 35 40 45 Val Asp Glu Thr Gly Met Gly Val Tyr Glu His Lys Val Ala Lys Asn 50 55 60 Gln Gly Lys Ser Lys Gly Val Trp Tyr Asn Leu His Asn Lys Lys Ser 65 70 75 80 Ile Gly Ile Leu Asn Ile Asp Glu Arg Thr Gly Met Ile Glu Ile Ala 85 90 95 Lys Pro Ile Gly Val Val Gly Ala Val Thr Pro Thr Thr Asn Pro Ile 100 105 110 Val Thr Pro Met Ser Asn Ile Ile Phe Ala Leu Lys Thr Cys Asn Ala 115 120 125 Ile Ile Ile Ala Pro His Pro Arg Ser Lys Lys Cys Ser Ala His Ala 130 135 140 Val Arg Leu Ile Lys Glu Ala Ile Ala Pro Phe Asn Val Pro Glu Gly 145 150 155 160 Met Val Gln Ile Ile Glu Glu Pro Ser Ile Glu Lys Thr Gln Glu Leu 165 170 175 Met Gly Ala Val Asp Val Val Val Ala Thr Gly Gly Met Gly Met Val 180 185 190 Lys Ser Ala Tyr Ser Ser Gly Lys Pro Ser Phe Gly Val Gly Ala Gly 195 200 205 Asn Val Gln Val Ile Val Asp Ser Asn Ile Asp Phe Glu Ala Ala Ala 210 215 220 Glu Lys Ile Ile Thr Gly Arg Ala Phe Asp Asn Gly Ile Ile Cys Ser 225 230 235 240 Gly Glu Gln Ser Ile Ile Tyr Asn Glu Ala Asp Lys Glu Ala Val Phe 245 250 255 Thr Ala Phe Arg Asn His Gly Ala Tyr Phe Cys Asp Glu Ala Glu Gly 260 265 270 Asp Arg Ala Arg Ala Ala Ile Phe Glu Asn Gly Ala Ile Ala Lys Asp 275 280 285 Val Val Gly Gln Ser Val Ala Phe Ile Ala Lys Lys Ala Asn Ile Asn 290 295 300 Ile Pro Glu Gly Thr Arg Ile Leu Val Val Glu Ala Arg Gly Val Gly 305 310 315 320 Ala Glu Asp Val Ile Cys Lys Glu Lys Met Cys Pro Val Met Cys Ala 325 330 335 Leu Ser Tyr Lys His Phe Glu Glu Gly Val Glu Ile Ala Arg Thr Asn 340 345 350 Leu Ala Asn Glu Gly Asn Gly His Thr Cys Ala Ile His Ser Asn Asn 355 360 365 Gln Ala His Ile Ile Leu Ala Gly Ser Glu Leu Thr Val Ser Arg Ile 370 375 380 Val Val Asn Ala Pro Ser Ala Thr Thr Ala Gly Gly His Ile Gln Asn 385 390 395 400 Gly Leu Ala Val Thr Asn Thr Leu Gly Cys Gly Ser Trp Gly Asn Asn 405 410 415 Ser Ile Ser Glu Asn Phe Thr Tyr Lys His Leu Leu Asn Ile Ser Arg 420 425 430 Ile Ala Pro Leu Asn Ser Ser Ile His Ile Pro Asp Asp Lys Glu Ile 435 440 445 Trp Glu Leu 450 <210> 12 <211> 1356 <212> DNA <213> Porphyromonas gingivalis <400> 12 atggaaataa aagagatggt gtcgttggca aggaaagctc agaaggaata tcaagcgacc 60 cataatcaag aagcagttga taacatttgc cgagctgcag caaaagtgat ttatgaaaat 120 gcagctatac tggctcgcga agcagtagac gaaaccggca tgggcgtata tgaacataaa 180 gtggccaaga atcaggggaa atccaaaggc gtctggtaca atttgcacaa taaaaaatcg 240 atcggtatct taaatataga cgagagaacc gggatgatcg agatagcaaa acctatcggg 300 gttgttggag ccgtaacccc gacgacaaac ccgattgtga ctccaatgag caacatcatt 360 tttgccctta agacatgcaa tgccattatt atcgccccac atcccagatc caaaaaatgc 420 tcagcacatg cagttcgtct gataaaggaa gcaatcgctc cgtttaatgt cccggaggga 480 atggttcaga tcattgaaga gcccagcatc gagaaaactc aggaactaat gggcgccgtg 540 gatgtggtag ttgcgacggg tggtatgggt atggtgaaat ctgcatattc ttcagggaag 600 ccttcttttg gtgtaggagc cggtaacgtt caagtgatcg tggatagtaa tatcgatttt 660 gaagctgcgg cagaaaaaat tatcaccggc cgtgctttcg acaatgggat catctgttca 720 ggcgaacaga gtatcatcta caacgaagct gacaaggaag ctgtcttcac agccttccgc 780 aaccatggtg catatttttg tgatgaagcg gagggagatc gggcccgtgc tgcgattttt 840 gagaatggcg ccatcgcgaa agatgtagtc ggccagagcg ttgcctttat cgcgaagaaa 900 gcaaatatca atataccgga gggtacccgt attctggttg ttgaagctcg cggcgtcgga 960 gcagaggatg tcatatgtaa ggaaaaaatg tgtccagtta tgtgcgcctt aagctacaag 1020 cacttcgagg aaggtgtaga aatcgcacgt acgaacttgg ccaacgaagg taacggccat 1080 acctgtgcga tccattccaa caatcaggcg catatcatac tggcaggttc agaactgacg 1140 gtttcgcgga tcgtggtcaa tgcgccgagt gccactacag caggcggtca catccaaaat 1200 ggtctggcag tgacaaatac gctcggatgc gggagttggg gtaataactc tatctccgag 1260 aactttactt ataaacacct gttaaacatt agccgcatag cgccgcttaa ttcaagcatt 1320 cacattcctg atgacaaaga gatctgggaa ctctaa 1356 <210> 13 <211> 371 <212> PRT <213> Porphyromonas gingivalis <400> 13 Met Gln Leu Phe Lys Leu Lys Ser Val Thr His His Phe Asp Thr Phe 1 5 10 15 Ala Glu Phe Ala Lys Glu Phe Cys Leu Gly Glu Arg Asp Leu Val Ile 20 25 30 Thr Asn Glu Phe Ile Tyr Glu Pro Tyr Met Lys Ala Cys Gln Leu Pro 35 40 45 Cys His Phe Val Met Gln Glu Lys Tyr Gly Gln Gly Glu Pro Ser Asp 50 55 60 Glu Met Met Asn Asn Ile Leu Ala Asp Ile Arg Asn Ile Gln Phe Asp 65 70 75 80 Arg Val Ile Gly Ile Gly Gly Gly Thr Val Ile Asp Ile Ser Lys Leu 85 90 95 Phe Val Leu Lys Gly Leu Asn Asp Val Leu Asp Ala Phe Asp Arg Lys 100 105 110 Ile Pro Leu Ile Lys Glu Lys Glu Leu Ile Ile Val Pro Thr Thr Cys 115 120 125 Gly Thr Gly Ser Glu Val Thr Asn Ile Ser Ile Ala Glu Ile Lys Ser 130 135 140 Arg His Thr Lys Met Gly Leu Ala Asp Asp Ala Ile Val Ala Asp His 145 150 155 160 Ala Ile Ile Ile Pro Glu Leu Leu Lys Ser Leu Pro Phe His Phe Tyr 165 170 175 Ala Cys Ser Ala Ile Asp Ala Leu Ile His Ala Ile Glu Ser Tyr Val 180 185 190 Ser Pro Lys Ala Ser Pro Tyr Ser Arg Leu Phe Ser Glu Ala Ala Trp 195 200 205 Asp Ile Ile Leu Glu Val Phe Lys Lys Ile Ala Glu His Gly Pro Glu 210 215 220 Tyr Arg Phe Glu Lys Leu Gly Glu Met Ile Met Ala Ser Asn Tyr Ala 225 230 235 240 Gly Ile Ala Phe Gly Asn Ala Gly Val Gly Ala Val His Ala Leu Ser 245 250 255 Tyr Pro Leu Gly Gly Asn Tyr His Val Pro His Gly Glu Ala Asn Tyr 260 265 270 Gln Phe Phe Thr Glu Val Phe Lys Val Tyr Gln Lys Lys Asn Pro Phe 275 280 285 Gly Tyr Ile Val Glu Leu Asn Trp Lys Leu Ser Lys Ile Leu Asn Cys 290 295 300 Gln Pro Glu Tyr Val Tyr Pro Lys Leu Asp Glu Leu Leu Gly Cys Leu 305 310 315 320 Leu Thr Lys Lys Pro Leu His Glu Tyr Gly Met Lys Asp Glu Glu Val 325 330 335 Arg Gly Phe Ala Glu Ser Val Leu Lys Thr Gln Gln Arg Leu Leu Ala 340 345 350 Asn Asn Tyr Val Glu Leu Thr Val Asp Glu Ile Glu Gly Ile Tyr Arg 355 360 365 Arg Leu Tyr 370 <210> 14 <211> 1116 <212> DNA <213> Porphyromonas gingivalis <400> 14 atgcaactgt tcaaactgaa atcagtcaca catcacttcg atactttcgc ggaatttgcc 60 aaagagttct gtcttggaga acgtgattta gtaattacca acgaattcat ttacgaaccg 120 tatatgaagg catgtcagtt gccctgccat tttgttatgc aggagaaata tgggcaaggc 180 gagccatctg acgagatgat gaataacatc ttggcagaca tccgtaatat ccagtttgac 240 cgcgtgatcg gtattggggg tggtacggtt attgacatct cgaaattatt tgtgctgaaa 300 ggactaaatg atgtgctcga tgcgttcgat cgcaagatac cgctgattaa agagaaagaa 360 ctgatcattg tgcccaccac atgcgggacg ggtagcgagg tgacgaatat ttcgatcgcg 420 gagatcaaaa gccgtcatac caaaatgggt ttggctgacg atgctattgt tgcagaccac 480 gcgatcatca taccagagct tctgaaaagc ctgccgttcc atttttatgc atgcagtgca 540 atagatgctc tgatccatgc catcgagtca tatgtttctc ctaaagccag tccatattct 600 cgtctgttca gtgaggcggc atgggatatt atcctggagg tattcaagaa aatagccgaa 660 cacggccctg aataccgctt tgagaagctg ggagaaatga tcatggcctc caactatgct 720 ggtatagcct tcgggaatgc aggcgtgggt gccgttcacg ctctaagcta tccattggga 780 ggcaattatc atgtgccgca tggcgaggct aactatcagt tttttacaga ggtctttaaa 840 gtataccaaa agaaaaatcc tttcggctat atagtcgaac tcaactggaa gctgtccaag 900 attctgaact gtcagcctga atacgtctat ccgaaactgg atgagttact cggctgtctt 960 ctgaccaaaa aaccgctgca cgaatacggc atgaaagatg aagaggtacg tggatttgcg 1020 gaatcagtgc ttaagactca gcagcggttg ctcgcgaata attatgttga gcttactgtt 1080 gatgaaattg aaggtatcta cagacgactg tactaa 1116 <210> 15 <211> 431 <212> PRT <213> Porphyromonas gingivalis <400> 15 Met Lys Asp Val Leu Ala Glu Tyr Ala Ser Arg Ile Val Ser Ala Glu 1 5 10 15 Glu Ala Val Lys His Ile Lys Asn Gly Glu Arg Val Ala Leu Ser His 20 25 30 Ala Ala Gly Val Pro Gln Ser Cys Val Asp Ala Leu Val Gln Gln Ala 35 40 45 Asp Leu Phe Gln Asn Val Glu Ile Tyr His Met Leu Cys Leu Gly Glu 50 55 60 Gly Lys Tyr Met Ala Pro Glu Met Ala Pro His Phe Arg His Ile Thr 65 70 75 80 Asn Phe Val Gly Gly Asn Ser Arg Lys Ala Val Glu Glu Asn Arg Ala 85 90 95 Asp Phe Ile Pro Val Phe Phe Tyr Glu Val Pro Ser Met Ile Arg Lys 100 105 110 Asp Ile Leu His Ile Asp Val Ala Ile Val Gln Leu Ser Met Pro Asp 115 120 125 Glu Asn Gly Tyr Cys Ser Phe Gly Val Ser Cys Asp Tyr Ser Lys Pro 130 135 140 Ala Ala Glu Ser Ala His Leu Val Ile Gly Glu Ile Asn Arg Gln Met 145 150 155 160 Pro Tyr Val His Gly Asp Asn Leu Ile His Ile Ser Lys Leu Asp Tyr 165 170 175 Ile Val Met Ala Asp Tyr Pro Ile Tyr Ser Leu Ala Lys Pro Lys Ile 180 185 190 Gly Glu Val Glu Glu Ala Ile Gly Arg Asn Cys Ala Glu Leu Ile Glu 195 200 205 Asp Gly Ala Thr Leu Gln Leu Gly Ile Gly Ala Ile Pro Asp Ala Ala 210 215 220 Leu Leu Phe Leu Lys Asp Lys Lys Asp Leu Gly Ile His Thr Glu Met 225 230 235 240 Phe Ser Asp Gly Val Val Glu Leu Val Arg Ser Gly Val Ile Thr Gly 245 250 255 Lys Lys Lys Thr Leu His Pro Gly Lys Met Val Ala Thr Phe Leu Met 260 265 270 Gly Ser Glu Asp Val Tyr His Phe Ile Asp Lys Asn Pro Asp Val Glu 275 280 285 Leu Tyr Pro Val Asp Tyr Val Asn Asp Pro Arg Val Ile Ala Gln Asn 290 295 300 Asp Asn Met Val Ser Ile Asn Ser Cys Ile Glu Ile Asp Leu Met Gly 305 310 315 320 Gln Val Val Ser Glu Cys Ile Gly Ser Lys Gln Phe Ser Gly Thr Gly 325 330 335 Gly Gln Val Asp Tyr Val Arg Gly Ala Ala Trp Ser Lys Asn Gly Lys 340 345 350 Ser Ile Met Ala Ile Pro Ser Thr Ala Lys Asn Gly Thr Ala Ser Arg 355 360 365 Ile Val Pro Ile Ile Ala Glu Gly Ala Ala Val Thr Thr Leu Arg Asn 370 375 380 Glu Val Asp Tyr Val Val Thr Glu Tyr Gly Ile Ala Gln Leu Lys Gly 385 390 395 400 Lys Ser Leu Arg Gln Arg Ala Glu Ala Leu Ile Ala Ile Ala His Pro 405 410 415 Asp Phe Arg Glu Glu Leu Thr Lys His Leu Arg Lys Arg Phe Gly 420 425 430 <210> 16 <211> 1296 <212> DNA <213> Porphyromonas gingivalis <400> 16 atgaaagacg tgttagcgga atatgcctcc cgaattgttt cggccgaaga ggcagtcaaa 60 catatcaaaa atggagagcg tgtcgcttta tcacatgctg ccggagttcc tcagagttgt 120 gttgacgcac tggtgcaaca ggcggacctg tttcagaatg tggagattta ccacatgctg 180 tgtctcggcg aaggaaaata tatggcacct gaaatggccc ctcacttccg gcacataacc 240 aattttgttg gtggtaactc tcgtaaagca gtggaggaaa atagagccga cttcattccg 300 gtattctttt atgaagtgcc atcaatgatt cggaaagata tccttcatat agatgtggcc 360 attgtccaac tctcaatgcc agatgagaat ggttactgca gctttggcgt atcttgcgat 420 tatagcaaac cggcggcgga atcggcgcat ttagttattg gggaaatcaa ccgtcagatg 480 ccatatgtgc atggtgacaa cttgattcac atatcgaagt tggattacat cgtgatggcg 540 gattacccaa tttattctct ggcgaagccc aaaatcggag aagtagagga agctatcggc 600 cgtaactgtg ccgagcttat tgaagatggt gccaccctac agctgggtat cggcgcgatt 660 ccggatgcag ctctgctgtt tctgaaggac aaaaaagatc tggggattca tactgaaatg 720 ttctccgatg gcgttgttga actggtgcgc agtggtgtaa ttactggaaa aaaaaagaca 780 ttgcatcccg gtaagatggt cgcgacgttt cttatgggat cagaagacgt gtatcatttc 840 atcgacaaga atccggatgt ggaactgtat ccggttgatt acgtcaatga tccgagggtt 900 atcgctcaga atgataatat ggtcagcatc aatagctgta tcgagatcga tctaatgggc 960 caagtggtga gcgagtgcat aggctccaaa cagtttagtg gcaccggggg tcaagtagat 1020 tatgtccgcg gggcagcttg gtctaaaaac ggcaaaagca tcatggcaat tccctcaaca 1080 gccaaaaacg gtactgcatc tcggatagtt cctataattg cagagggcgc tgctgtaaca 1140 accctccgca acgaagtcga ctacgttgtt acggaatatg ggatagcaca gttaaaaggt 1200 aagagtttgc gtcagcgcgc agaagctctt attgcgatag cccacccgga ctttagagag 1260 gaactgacga agcatctgcg caaacgtttt ggttaa 1296 <210> 17 <211> 858 <212> PRT <213> Clostridium acetobutylicum <400> 17 Met Lys Val Thr Asn Gln Lys Glu Leu Lys Gln Lys Leu Asn Glu Leu 1 5 10 15 Arg Glu Ala Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val Asp 20 25 30 Lys Ile Phe Lys Gln Cys Ala Ile Ala Ala Ala Lys Glu Arg Ile Asn 35 40 45 Leu Ala Lys Leu Ala Val Glu Glu Thr Gly Ile Gly Leu Val Glu Asp 50 55 60 Lys Ile Ile Lys Asn His Phe Ala Ala Glu Tyr Ile Tyr Asn Lys Tyr 65 70 75 80 Lys Asn Glu Lys Thr Cys Gly Ile Ile Asp His Asp Asp Ser Leu Gly 85 90 95 Ile Thr Lys Val Ala Glu Pro Ile Gly Ile Val Ala Ala Ile Val Pro 100 105 110 Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu 115 120 125 Lys Thr Arg Asn Ala Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys 130 135 140 Ser Thr Ile Ala Ala Ala Lys Leu Ile Leu Asp Ala Ala Val Lys Ala 145 150 155 160 Gly Ala Pro Lys Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu 165 170 175 Leu Ser Gln Asp Leu Met Ser Glu Ala Asp Ile Ile Leu Ala Thr Gly 180 185 190 Gly Pro Ser Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile 195 200 205 Gly Val Gly Ala Gly Asn Thr Pro Ala Ile Ile Asp Glu Ser Ala Asp 210 215 220 Ile Asp Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn 225 230 235 240 Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Leu Val Met Asn Ser Ile 245 250 255 Tyr Glu Lys Val Lys Glu Glu Phe Val Lys Arg Gly Ser Tyr Ile Leu 260 265 270 Asn Gln Asn Glu Ile Ala Lys Ile Lys Glu Thr Met Phe Lys Asn Gly 275 280 285 Ala Ile Asn Ala Asp Ile Val Gly Lys Ser Ala Tyr Ile Ile Ala Lys 290 295 300 Met Ala Gly Ile Glu Val Pro Gln Thr Thr Lys Ile Leu Ile Gly Glu 305 310 315 320 Val Gln Ser Val Glu Lys Ser Glu Leu Phe Ser His Glu Lys Leu Ser 325 330 335 Pro Val Leu Ala Met Tyr Lys Val Lys Asp Phe Asp Glu Ala Leu Lys 340 345 350 Lys Ala Gln Arg Leu Ile Glu Leu Gly Gly Ser Gly His Thr Ser Ser 355 360 365 Leu Tyr Ile Asp Ser Gln Asn Asn Lys Asp Lys Val Lys Glu Phe Gly 370 375 380 Leu Ala Met Lys Thr Ser Arg Thr Phe Ile Asn Met Pro Ser Ser Gln 385 390 395 400 Gly Ala Ser Gly Asp Leu Tyr Asn Phe Ala Ile Ala Pro Ser Phe Thr 405 410 415 Leu Gly Cys Gly Thr Trp Gly Gly Asn Ser Val Ser Gln Asn Val Glu 420 425 430 Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn 435 440 445 Met Leu Trp Phe Lys Val Pro Gln Lys Ile Tyr Phe Lys Tyr Gly Cys 450 455 460 Leu Arg Phe Ala Leu Lys Glu Leu Lys Asp Met Asn Lys Lys Arg Ala 465 470 475 480 Phe Ile Val Thr Asp Lys Asp Leu Phe Lys Leu Gly Tyr Val Asn Lys 485 490 495 Ile Thr Lys Val Leu Asp Glu Ile Asp Ile Lys Tyr Ser Ile Phe Thr 500 505 510 Asp Ile Lys Ser Asp Pro Thr Ile Asp Ser Val Lys Lys Gly Ala Lys 515 520 525 Glu Met Leu Asn Phe Glu Pro Asp Thr Ile Ile Ser Ile Gly Gly Gly 530 535 540 Ser Pro Met Asp Ala Ala Lys Val Met His Leu Leu Tyr Glu Tyr Pro 545 550 555 560 Glu Ala Glu Ile Glu Asn Leu Ala Ile Asn Phe Met Asp Ile Arg Lys 565 570 575 Arg Ile Cys Asn Phe Pro Lys Leu Gly Thr Lys Ala Ile Ser Val Ala 580 585 590 Ile Pro Thr Thr Ala Gly Thr Gly Ser Glu Ala Thr Pro Phe Ala Val 595 600 605 Ile Thr Asn Asp Glu Thr Gly Met Lys Tyr Pro Leu Thr Ser Tyr Glu 610 615 620 Leu Thr Pro Asn Met Ala Ile Ile Asp Thr Glu Leu Met Leu Asn Met 625 630 635 640 Pro Arg Lys Leu Thr Ala Ala Thr Gly Ile Asp Ala Leu Val His Ala 645 650 655 Ile Glu Ala Tyr Val Ser Val Met Ala Thr Asp Tyr Thr Asp Glu Leu 660 665 670 Ala Leu Arg Ala Ile Lys Met Ile Phe Lys Tyr Leu Pro Arg Ala Tyr 675 680 685 Lys Asn Gly Thr Asn Asp Ile Glu Ala Arg Glu Lys Met Ala His Ala 690 695 700 Ser Asn Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly Val Cys 705 710 715 720 His Ser Met Ala His Lys Leu Gly Ala Met His His Val Pro His Gly 725 730 735 Ile Ala Cys Ala Val Leu Ile Glu Glu Val Ile Lys Tyr Asn Ala Thr 740 745 750 Asp Cys Pro Thr Lys Gln Thr Ala Phe Pro Gln Tyr Lys Ser Pro Asn 755 760 765 Ala Lys Arg Lys Tyr Ala Glu Ile Ala Glu Tyr Leu Asn Leu Lys Gly 770 775 780 Thr Ser Asp Thr Glu Lys Val Thr Ala Leu Ile Glu Ala Ile Ser Lys 785 790 795 800 Leu Lys Ile Asp Leu Ser Ile Pro Gln Asn Ile Ser Ala Ala Gly Ile 805 810 815 Asn Lys Lys Asp Phe Tyr Asn Thr Leu Asp Lys Met Ser Glu Leu Ala 820 825 830 Phe Asp Asp Gln Cys Thr Thr Ala Asn Pro Arg Tyr Pro Leu Ile Ser 835 840 845 Glu Leu Lys Asp Ile Tyr Ile Lys Ser Phe 850 855 <210> 18 <211> 2577 <212> DNA <213> Clostridium acetobutylicum <400> 18 atgaaagtca ccaaccagaa agagctgaaa cagaaactga acgaactgcg tgaagcccag 60 aagaagttcg ctacgtacac ccaggaacag gtggacaaaa tcttcaaaca gtgcgcgatt 120 gctgctgcaa aagaacgtat caacctggct aaactggccg tggaagagac gggcattggt 180 ctggtggaag acaagatcat caaaaaccac tttgcggcgg aatacatcta caacaagtac 240 aaaaacgaga aaacttgcgg catcatcgac cacgatgatt ccctgggcat caccaaagtg 300 gcagaaccta tcggtattgt tgcagcaatt gtaccgacta ctaacccgac ttctaccgct 360 attttcaagt ctctgatttc tctgaaaacc cgcaacgcga ttttcttctc tccgcaccca 420 cgtgcgaaaa aatccaccat cgctgccgcc aaactgatcc tggacgcggc agttaaagcg 480 ggtgctccga aaaatatcat tggttggatc gatgaaccgt ctatcgaact gagccaggat 540 ctgatgtccg aagcagacat tatcctggca accggcggtc cgtctatggt aaaagccgcc 600 tactcttctg gcaaaccggc aattggtgtt ggtgctggta acacgccggc gattatcgac 660 gagtccgcag acatcgatat ggcagtttcc tctatcattc tgtccaaaac ctacgataac 720 ggcgtgatct gcgcgagcga acagtccatc ctggttatga attctatcta tgaaaaggtc 780 aaggaagaat ttgttaagcg tggcagctac atcctgaacc agaacgagat cgcgaaaatc 840 aaagaaacta tgttcaaaaa cggtgccatc aatgccgaca tcgtcggcaa atctgcttac 900 attattgcca aaatggctgg tatcgaagtg ccgcagacca cgaagatcct gatcggtgag 960 gtacagagcg ttgaaaagtc tgaactgttc tctcatgaaa aactgtcccc ggtcctggct 1020 atgtacaaag taaaagactt cgacgaagca ctgaaaaaag cgcaacgtct gatcgagctg 1080 ggtggtagcg gccacacctc tagcctgtac atcgacagcc agaacaacaa agataaagtt 1140 aaagaattcg gcctggcaat gaaaaccagc cgcaccttta ttaacatgcc ttctagccaa 1200 ggtgcttctg gcgacctgta taacttcgct attgcgcctt cctttaccct gggttgcggt 1260 acctggggcg gtaacagcgt ttcccaaaac gttgaaccga aacacctgct gaacattaaa 1320 tctgtagcag aacgccgtga gaacatgctg tggtttaaag ttccgcagaa aatctacttc 1380 aagtacggtt gtctgcgctt cgctctgaaa gaactgaagg atatgaacaa gaaacgtgcg 1440 ttcatcgtga ctgataaaga tctgttcaaa ctgggctacg ttaacaaaat cactaaagta 1500 ctggacgaaa tcgatattaa gtattccatc tttaccgaca tcaaatctga cccgaccatc 1560 gattccgtaa aaaagggtgc taaggaaatg ctgaacttcg aaccggacac tattatcagc 1620 atcggcggtg gctctccgat ggatgcagca aaagtgatgc atctgctgta cgaatacccg 1680 gaagcggaaa tcgaaaacct ggcgatcaat ttcatggaca tccgtaaacg tatctgcaat 1740 tttccgaagc tgggtacgaa agccatttcc gttgcgattc cgactaccgc gggtactggt 1800 tctgaagcga ccccgttcgc tgttattact aacgatgaaa ctggtatgaa atacccactg 1860 acgagctatg agctgacccc aaacatggca atcattgata ccgagctgat gctgaatatg 1920 ccgcgtaaac tgaccgcggc gactggcatc gacgccctgg ttcacgcgat cgaagcttat 1980 gtttctgtca tggccaccga ttatacggac gaactggctc tgcgtgctat caaaatgatt 2040 ttcaaatatc tgcctcgcgc gtacaagaac ggcaccaacg atattgaggc tcgtgaaaaa 2100 atggcacacg ccagcaacat cgcaggcatg gcattcgcta acgcttttct gggcgtatgc 2160 cattccatgg ctcataaact gggtgcaatg caccacgttc cacacggcat cgcgtgtgcg 2220 gtgctgatcg aagaggtgat caaatacaac gctactgact gtccgactaa acaaaccgcg 2280 tttccgcagt acaaatcccc aaatgcgaaa cgtaaatatg cggagatcgc cgaatatctg 2340 aacctgaaag gcacctccga caccgaaaaa gtgaccgctc tgattgaagc catcagcaaa 2400 ctgaaaattg acctgtctat cccgcagaac atcagcgcgg caggtatcaa caaaaaagat 2460 ttctataaca ccctggataa aatgagcgag ctggcgttcg atgaccagtg tacgaccgca 2520 aacccgcgct acccgctgat ctccgaactg aaagacattt atattaaatc cttctaa 2577 <210> 19 <211> 468 <212> PRT <213> Clostridium beijerinckii <400> 19 Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Val Lys 1 5 10 15 Thr Asn Gly Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser 20 25 30 Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Ser Ala Val 35 40 45 His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu 50 55 60 Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Gln Asn Lys Glu Val 65 70 75 80 Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp 85 90 95 Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu 100 105 110 Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val 115 120 125 Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn 130 135 140 Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly 145 150 155 160 Asn Ala Val Val Phe Asn Gly His Pro Cys Ala Lys Lys Cys Val Ala 165 170 175 Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro 180 185 190 Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp 195 200 205 Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly 210 215 220 Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly 225 230 235 240 Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile 245 250 255 Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn 260 265 270 Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala 275 280 285 Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn 290 295 300 Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn 305 310 315 320 Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala 325 330 335 Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys 340 345 350 Cys Ile Ile Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu 355 360 365 Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu 370 375 380 Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala 385 390 395 400 Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu 405 410 415 Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val 420 425 430 Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr 435 440 445 Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys 450 455 460 Val Leu Ala Gly 465 <210> 20 <211> 1407 <212> DNA <213> Clostridium beijerinckii <400> 20 atgaataagg atacgttgat cccgaccacc aaggatctga aggtcaagac caatggcgag 60 aacatcaatc ttaaaaacta caaagacaac agctcctgtt tcggagtgtt tgaaaatgtg 120 gaaaacgcga tctcctcagc agtacacgcg caaaagatcc tctctttgca ctacactaag 180 gaacagcgtg aaaagattat cacggagatc cgcaaagcgg cactgcagaa caaagaggtc 240 ctggctacaa tgatcttgga ggagacacat atgggtcgct acgaggacaa gatcctcaag 300 cacgagcttg ttgctaagta cacccccggc accgaggatc ttaccaccac cgcctggtct 360 ggcgataatg gactgaccgt tgtggaaatg tccccctacg gcgttatcgg ggcaattacc 420 ccaagcacaa acccaaccga aaccgtgatt tgtaactcga tcggaatgat cgccgcaggt 480 aacgctgtgg ttttcaacgg ccacccatgc gcaaagaagt gcgttgcatt tgccgtggag 540 atgatcaaca aggcaattat ctcatgcggt ggtcctgaaa acctcgtcac taccattaag 600 aatccaacta tggagtcgct tgatgctatc attaagcacc catcgatcaa gcttctctgt 660 ggaactggcg gccctggcat ggtcaaaacg ctcctgaaca gcgggaaaaa ggcgattgga 720 gccggtgcag gtaatccgcc cgtcatcgtg gacgatacgg cagatattga gaaggccggt 780 cgttccatca tcgaaggctg ctcatttgat aacaacctgc cgtgcattgc tgagaaagaa 840 gttttcgttt tcgagaacgt tgccgatgac cttatttcca atatgttgaa gaataatgca 900 gtgatcatca acgaagacca agtttccaaa ctgatcgatc tcgtccttca gaaaaacaac 960 gagactcagg aatatttcat taacaagaag tgggtgggca aagacgcaaa gctgttcttg 1020 gatgagattg acgtggagag cccttccaac gtcaagtgca ttatctgtga agtcaacgct 1080 aaccatcctt tcgtgatgac ggaattgatg atgccaatcc tgccgattgt tcgagtaaaa 1140 gacattgacg aagctatcaa gtacgcgaaa atcgccgaac agaaccgcaa gcactctgct 1200 tatatctact ctaagaacat tgacaatctg aaccggtttg aacgggagat cgacactacc 1260 atctttgtca aaaacgcgaa atccttcgct ggcgtgggct atgaagctga gggattcacc 1320 accttcacca ttgcggggag caccggtgaa ggcatcactt ctgcccgcaa cttcacccgc 1380 cagcgccgtt gcgtactcgc cggttaa 1407 <210> 21 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> ldhA KO primer_up <400> 21 atgaaactcg ccgtttatag cacaaaacag tacgacaaga agtacctgca taggtgacac 60 tatagaacgc g 71 <210> 22 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> ldhA KO primer_do <400> 22 ttaaaccagt tcgttcgggc aggtttcgcc tttttccaga ttgcttaagt tagtggatct 60 gatgggtacc 70 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_up <400> 23 tacactaagc atagttgttg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_do <400> 24 ctttcttcat tgtggttctc 20 <210> 25 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> adhE KO primer <400> 25 atggctgtta ctaatgtcgc tgaacttaac gcactcgtag agcgtgtaaa taggtgacac 60 tatagaacgc g 71 <210> 26 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> adhE KO primer <400> 26 ttaagcggat tttttcgctt ttttctcagc tttagccgga gcggcttctt tagtggatct 60 gatgggtacc 70 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer-up <400> 27 caccgcactg actatactct 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer-do <400> 28 gatgaaggct aatgctgtcg 20 <210> 29 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> mdh KO primer_up <400> 29 atgaaagtcg cagtcctcgg cgctgctggc ggtattggcc aggcgcttgc taggtgacac 60 tatagaacgc g 71 <210> 30 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> mdh KO primer_do <400> 30 ttacttatta acgaactctt cgcccagggc gatatctttc ttcagcgtat tagtggatct 60 gatgggtacc 70 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_up <400> 31 ggttcctgat tacggcaatt 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_do <400> 32 attcaggaat atccggcaac 20 <210> 33 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 gctagaattc atgaacttac atgaatatca gg 32 <210> 34 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 gcaaggtacc ttatttcaga acagttttca g 31 <210> 35 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 gaaaactgtt ctgaaataag tcacacagga aacagaattc atggaaataa aagagatg 58 <210> 36 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 ttcagtttga acagttgcat gaattctgtt tcctgtgtga ttagagttcc cagatctc 58 <210> 37 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 aagagatctg ggaactctaa tcacacagga aacagaattc atgcaactgt tcaaactg 58 <210> 38 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 cgactctaga ggatccccgg ttagtacagt cgtctgtaga taccttc 47 <110> Samsung Electronics Co. Ltd. <120> Microorganism capable of producing 1,4-BDO and method of          producing 1,4-BDO using the same <130> PN100757 <160> 38 <170> Kopatentin 2.0 <210> 1 <211> 329 <212> PRT <213> E. coli <400> 1 Met Lys Leu Ala Val Tyr Ser Thr Lys Gln Tyr Asp Lys Lys Tyr Leu   1 5 10 15 Gln Gln Val Asn Glu Ser Phe Gly Phe Glu Leu Glu Phe Phe Asp Phe              20 25 30 Leu Leu Thr Glu Lys Thr Ala Lys Thr Ala Asn Gly Cys Glu Ala Val          35 40 45 Cys Ile Phe Val Asn Asp Asp Gly Ser Arg Pro Val Leu Glu Glu Leu      50 55 60 Lys Lys His Gly Val Lys Tyr Ile Ala Leu Arg Cys Ala Gly Phe Asn  65 70 75 80 Asn Val Asp Leu Asp Ala Ala Lys Glu Leu Gly Leu Lys Val Val Arg                  85 90 95 Val Pro Ala Tyr Asp Pro Glu Ala Val Ala Glu His Ala Ile Gly Met             100 105 110 Met Met Thr Leu Asn Arg Arg Ile His Arg Ala Tyr Gln Arg Thr Arg         115 120 125 Asp Ala Asn Phe Ser Leu Glu Gly Leu Thr Gly Phe Thr Met Tyr Gly     130 135 140 Lys Thr Ala Gly Val Ile Gly Thr Gly Lys Ile Gly Val Ala Met Leu 145 150 155 160 Arg Ile Leu Lys Gly Phe Gly Met Arg Leu Leu Ala Phe Asp Pro Tyr                 165 170 175 Pro Ser Ala Ala Leu Glu Leu Gly Val Glu Tyr Val Asp Leu Pro             180 185 190 Thr Leu Phe Ser Glu Ser Asp Val Ile Ser Leu His Cys Pro Leu Thr         195 200 205 Pro Glu Asn Tyr His Leu Leu Asn Glu Ala Ala Phe Asp Gln Met Lys     210 215 220 Asn Gly Val Met Ile Val Asn Thr Ser Arg Gly Ala Leu Ile Asp Ser 225 230 235 240 Gln Ala Ala Ile Glu Ala Leu Lys Asn Gln Lys Ile Gly Ser Leu Gly                 245 250 255 Met Asp Val Tyr Glu Asn Glu Arg Asp Leu Phe Phe Glu Asp Lys Ser             260 265 270 Asn Asp Val Ile Gln Asp Asp Val Phe Arg Arg Leu Ser Ala Cys His         275 280 285 Asn Val Leu Phe Thr Gly His Gln Ala Phe Leu Thr Ala Glu Ala Leu     290 295 300 Thr Ser Ile Ser Gln Thr Thr Leu Gln Asn Leu Ser Asn Leu Glu Lys 305 310 315 320 Gly Glu Thr Cys Pro Asn Glu Leu Val                 325 <210> 2 <211> 990 <212> DNA <213> E. coli <400> 2 atgaaactcg ccgtttatag cacaaaacag tacgacaaga agtacctgca acaggtgaac 60 gagtcctttg gctttgagct ggaatttttt gactttctgc tgacggaaaa aaccgctaaa 120 actgccaatg gctgcgaagc ggtatgtatt ttcgtaaacg atgacggcag ccgcccggtg 180 ctggaagagc tgaaaaagca cggcgttaaa tatatcgccc tgcgctgtgc cggtttcaat 240 aacgtcgacc ttgacgcggc aaaagaactg gggctgaaag tagtccgtgt tccagcctat 300 gatccagagg ccgttgctga acacgccatc ggtatgatga tgacgctgaa ccgccgtatt 360 caccgcgcgt atcagcgtac ccgtgacgct aacttctctc tggaaggtct gaccggcttt 420 actatgtatg gcaaaacggc aggcgttatc ggtaccggta aaatcggtgt ggcgatgctg 480 cgcattctga aaggttttgg tatgcgtctg ctggcgttcg atccgtatcc aagtgcagcg 540 gcgctggaac tcggtgtgga gtatgtcgat ctgccaaccc tgttctctga atcagacgtt 600 atctctctgc actgcccgct gacaccggaa aactaccatc tgttgaacga agccgccttc 660 gatcagatga aaaatggcgt gatgatcgtc aataccagtc gcggtgcatt gattgattct 720 caggcagcaa ttgaagcgct gaaaaatcag aaaattggtt cgttgggtat ggacgtgtat 780 gagaacgaac gcgatctatt ctttgaagat aaatccaacg acgtaattca ggatgacgta 840 ttccgtcgcc tgtctgcctg ccacaacgtg ctatttaccg ggcaccaggc attcctgaca 900 gcagaagctc tgaccagtat ttctcagact acgctgcaaa acttaagcaa tctggaaaaa 960 ggcgaaacct gcccgaacga actggtttaa 990 <210> 3 <211> 891 <212> PRT <213> E. coli <400> 3 Met Ala Val Thr Asn Val Ala Glu Leu Asn Ala Leu Val Glu Arg Val   1 5 10 15 Lys Lys Ala Gln Arg Glu Tyr Ala Ser Phe Thr Gln Glu Gln Val Asp              20 25 30 Lys Ile Phe Arg Ala Ala Ala Ala Ala Ala Asp Ala Arg Ile Pro          35 40 45 Leu Ala Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val Glu Asp      50 55 60 Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr  65 70 75 80 Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp Asp Thr Phe Gly                  85 90 95 Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys Gly Ile Val Pro             100 105 110 Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu         115 120 125 Lys Thr Arg Asn Ala Ile Ile Phe Ser Pro His Pro Arg Ala Lys Asp     130 135 140 Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala Ala Ile Ala Ala 145 150 155 160 Gly Ala Pro Lys Asp Leu Ile Gly Trp Ile Asp Gln Pro Ser Val Glu                 165 170 175 Leu Ser Asn Ala Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala             180 185 190 Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro         195 200 205 Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Ile Asp Glu Thr     210 215 220 Ala Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys Thr Phe 225 230 235 240 Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val Val Val Val Asp                 245 250 255 Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Thr His Gly Gly Tyr             260 265 270 Leu Leu Gln Gly Lys Glu Leu Lys Ala Val Gln Asp Val Ile Leu Lys         275 280 285 Asn Gly Ala Leu Asn Ala Ile Val Gly Gln Pro Ala Tyr Lys Ile     290 295 300 Ala Glu Leu Ala Gly Phe Ser Val Pro Glu Asn Thr Lys Ile Leu Ile 305 310 315 320 Gly Glu Val Thr Val Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys                 325 330 335 Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu Asp Ala             340 345 350 Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly His Thr         355 360 365 Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala Arg Val Ser Tyr     370 375 380 Phe Gly Gln Lys Met Lys Thr Ala Arg Ile Leu Ile Asn Thr Pro Ala 385 390 395 400 Ser Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser                 405 410 415 Leu Thr Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn             420 425 430 Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala         435 440 445 Glu Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg     450 455 460 Gly Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly His Lys 465 470 475 480 Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn Gly Tyr Ala                 485 490 495 Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly Val Glu Thr Glu Val             500 505 510 Phe Phe Glu Val Glu Ala Asp Pro Thr Leu Ser Ile Val Arg Lys Gly         515 520 525 Ala Glu Leu Ala Asn Ser Phe Lys Pro Asp Val Ile Ile Ala Leu Gly     530 535 540 Gly Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu 545 550 555 560 His Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile                 565 570 575 Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Met             580 585 590 Ile Ala Val Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr Pro Phe         595 600 605 Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu Ala Asp     610 615 620 Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp Ala Asn Leu Val Met 625 630 635 640 Asp Met Pro Lys Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala Val Thr                 645 650 655 His Ala Met Glu Ala Tyr Val Ser Val Leu Ala Ser Glu Phe Ser Asp             660 665 670 Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Tyr Leu Pro Ala         675 680 685 Ser Tyr His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg Val His     690 695 700 Ser Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu Gly 705 710 715 720 Val Cys His Ser Ala His Lys Leu Gly Ser Gln Phe His Ile Pro                 725 730 735 His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val Ile Arg Tyr Asn             740 745 750 Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe Ser Gln Tyr Asp Arg         755 760 765 Pro Gln Ala Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu Gly Leu     770 775 780 Ser Ala Pro Gly Asp Arg Thr Ala Ala Lys Ile Glu Lys Leu Leu Ala 785 790 795 800 Trp Leu Glu Thr Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile Arg                 805 810 815 Glu Ala Gly Val Glu Glu Ala Asp Phe Leu Ala Asn Val Asp Lys Leu             820 825 830 Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr         835 840 845 Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr Tyr Gly     850 855 860 Arg Asp Tyr Val Glu Gly Glu Glu Thr Ala Ala Lys Lys Glu Ala Ala Pro 865 870 875 880 Ala Lys Ala Glu Lys Lys Ala Lys Lys Ser Ala                 885 890 <210> 4 <211> 2676 <212> DNA <213> E. coli <400> 4 atggctgtta ctaatgtcgc tgaacttaac gcactcgtag agcgtgtaaa aaaagcccag 60 cgtgaatatg ccagtttcac tcaagagcaa gtagacaaaa tcttccgcgc cgccgctctg 120 gctgctgcag atgctcgaat cccactcgcg aaaatggccg ttgccgaatc cggcatgggt 180 atcgtcgaag ataaagtgat caaaaaccac tttgcttctg aatatatcta caacgcctat 240 aaagatgaaa aaacctgtgg tgttctgtct gaagacgaca cttttggtac catcactatc 300 gctgaaccaa tcggtattat ttgcggtatc gttccgacca ctaacccgac ttcaactgct 360 atcttcaaat cgctgatcag tctgaagacc cgtaacgcca ttatcttctc cccgcacccg 420 cgtgcaaaag atgccaccaa caaagcggct gatatcgttc tgcaggctgc tatcgctgcc 480 ggtgctccga aagatctgat cggctggatc gatcaacctt ctgttgaact gtctaacgca 540 ctgatgcacc acccagacat caacctgatc ctcgcgactg gtggtccggg catggttaaa 600 gccgcataca gctccggtaa accagctatc ggtgtaggcg cgggcaacac tccagttgtt 660 atcgatgaaa ctgctgatat caaacgtgca gttgcatctg tactgatgtc caaaaccttc 720 gacaacggcg taatctgtgc ttctgaacag tctgttgttg ttgttgactc tgtttatgac 780 gctgtacgtg aacgttttgc aacccacggc ggctatctgt tgcagggtaa agagctgaaa 840 gctgttcagg atgttatcct gaaaaacggt gcgctgaacg cggctatcgt tggtcagcca 900 gcctataaaa ttgctgaact ggcaggcttc tctgtaccag aaaacaccaa gattctgatc 960 ggtgaagtga ccgttgttga tgaaagcgaa ccgttcgcac atgaaaaact gtccccgact 1020 ctggcaatgt accgcgctaa agatttcgaa gacgcggtag aaaaagcaga gaaactggtt 1080 gt; cgcgtttctt acttcggtca gaaaatgaaa acggctcgta tcctgattaa caccccagcg 1200 tctcagggtg gtatcggtga cctgtataac ttcaaactcg caccttccct gactctgggt 1260 tgtggttctt ggggtggtaa ctccatctct gaaaacgttg gtccgaaaca cctgatcaac 1320 aagaaaaccg ttgctaagcg agctgaaaac atgttgtggc acaaacttcc gaaatctatc 1380 tacttccgcc gtggctccct gccaatcgcg ctggatgaag tgattactga tggccacaaa 1440 cgtgcgctca tcgtgactga ccgcttcctg ttcaacaatg gttatgctga tcagatcact 1500 tccgtactga aagcagcagg cgttgaaact gaagtcttct tcgaagtaga agcggacccg 1560 accctgagca tcgttcgtaa aggtgcagaa ctggcaaact ccttcaaacc agacgtgatt 1620 atcgcgctgg gtggtggttc cccgatggac gccgcgaaga tcatgtgggt tatgtacgaa 1680 catccggaaa ctcacttcga agagctggcg ctgcgcttta tggatatccg taaacgtatc 1740 tacaagttcc cgaaaatggg cgtgaaagcg aaaatgatcg ctgtcaccac cacttctggt 1800 acaggttctg aagtcactcc gtttgcggtt gtaactgacg acgctactgg tcagaaatat 1860 ccgctggcag actatgcgct gactccggat atggcgattg tcgacgccaa cctggttatg 1920 gacatgccga agtccctgtg tgctttcggt ggtctggacg cagtaactca cgccatggaa 1980 gcttatgttt ctgtactggc atctgagttc tctgatggtc aggctctgca ggcactgaaa 2040 ctgctgaaag aatatctgcc agcgtcctac cacgaagggt ctaaaaatcc ggtagcgcgt 2100 gaacgtgttc acagtgcagc gactatcgcg ggtatcgcgt ttgcgaacgc cttcctgggt 2160 gtatgtcact caatggcgca caaactgggt tcccagttcc atattccgca cggtctggca 2220 aacgccctgc tgatttgtaa cgttattcgc tacaatgcga acgacaaccc gaccaagcag 2280 actgcattca gccagtatga ccgtccgcag gctcgccgtc gttatgctga aattgccgac 2340 cacttgggtc tgagcgcacc gggcgaccgt actgctgcta agatcgagaa actgctggca 2400 tggctggaaa cgctgaaagc tgaactgggt attccgaaat ctatccgtga agctggcgtt 2460 caggaagcag acttcctggc gaacgtggat aaactgtctg aagatgcgtt cgatgaccag 2520 tgcaccggcg ctaacccgcg ttacccgctg atctccgagc tgaaacagat cctgctggat 2580 acctactacg gtcgtgatta tgtagaaggt gaaactgcag cgaaaaaaga agccgctccg 2640 gctaaagctg agaaaaaagc gaaaaaatcc gcttaa 2676 <210> 5 <211> 312 <212> PRT <213> E. coli <400> 5 Met Lys Val Ala Val Leu Gly Ala Ala Gly   1 5 10 15 Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu              20 25 30 Tyr Asp Ile Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His          35 40 45 Ile Pro Thr Ala Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr      50 55 60 Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala  65 70 75 80 Arg Lys Pro Gly Met Asp Arg Ser Asp Leu Phe Asn Val Asn Ala Gly                  85 90 95 Ile Val Lys Asn Leu Val Gln Gln Val Ser Lys Thr Cys Pro Lys Ala             100 105 110 Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala         115 120 125 Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe     130 135 140 Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu 145 150 155 160 Leu Lys Gly Lys Gln Pro Gly Glu Val Glu Val Pro Val Ile Gly Gly                 165 170 175 His Ser Gly Val Thr Ile Leu Pro Leu Leu Ser Gln Val Pro Gly Val             180 185 190 Ser Phe Thr Glu Gln Glu Val Ala Asp Leu Thr Lys Arg Ile Gln Asn         195 200 205 Ala Gly Thr Glu Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr     210 215 220 Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg 225 230 235 240 Ala Leu Gln Gly Glu Gln Gly Val Val Glu Cys Ala Tyr Val Glu Gly                 245 250 255 Asp Gly Gln Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys             260 265 270 Asn Gly Val Glu Glu Arg Lys Ser Ile Gly Thr Leu Ser Ala Phe Glu         275 280 285 Gln Ser Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala     290 295 300 Leu Gly Glu Glu Phe Val Asn Lys 305 310 <210> 6 <211> 939 <212> DNA <213> E. coli <400> 6 atgaaagtcg cagtcctcgg cgctgctggc ggtattggcc aggcgcttgc actactgtta 60 aaaacccaac tgccttcagg ttcagaactc tctctgtatg atatcgctcc agtgactccc 120 ggtgtggctg tcgatctgag ccatatccct actgctgtga aaatcaaagg tttttctggt 180 gaagatgcga ctccggcgct ggaaggcgca gatgtcgttc ttatctctgc aggtgtagcg 240 cgtaaaccgg gtatggatcg ttccgacctg tttaacgtta acgccggcat cgtgaaaaac 300 ctggtacagc aagtttcgaa aacctgcccg aaagcgtgca ttggtattat cactaacccg 360 gttaacacca cagttgcgat tgctgctgaa gtgctgaaaa aagccggtgt ttatgacaaa 420 aacaaactgt tcggcgttac cacgctggat atcattcgtt ccaacacctt tgttgcggaa 480 ctgaaaggca aacagccagg cgaagttgaa gtgccggtta ttggcggtca ctctggtgtt 540 accattctgc cgctgctgtc acaggttcct ggcgttagtt ttaccgagca ggaagtggct 600 gatctgacca aacgtatcca gaacgcaggt actgaagtgg ttgaagcgaa agccggtggc 660 gggtctgcaa ccctgtctat gggccaggca gctgcacgtt ttggtctgtc tctggtacgc 720 gcactgcagg gcgaacaagg cgttgtcgaa tgtgcctatg ttgaaggcga cggtcagtac 780 gcacgtttct tctctcaacc gctgctgctg ggtaaaaacg gcgtggaaga gcgtaaatct 840 atcggtaccc tgagcgcatt tgaacagagc gcactggaag gtatgctgga tacgctgaag 900 aaagatatcg ccctgggcga agagttcgtt aataagtaa 939 <210> 7 <211> 677 <212> PRT <213> E. coli <400> 7 Met Asn Leu His Glu Tyr Gln Ala Lys Gln Leu Phe Ala Arg Tyr Gly   1 5 10 15 Leu Pro Ala Pro Val Gly Tyr Ala Cys Thr Thr Pro Arg Glu Ala Glu              20 25 30 Glu Ala Ala Ser Lys Ile Gly Ala Gly Pro Trp Val Val Lys Cys Gln          35 40 45 Val His Ala Gly Gly Arg Gly Lys Ala Gly Gly Val Lys Val Val Asn      50 55 60 Ser Lys Glu Asp Ile Arg Ala Phe Ala Glu Asn Trp Leu Gly Lys Arg  65 70 75 80 Leu Val Thr Tyr Gln Thr Asp Ala Asn Gly Gln Pro Val Asn Gln Ile                  85 90 95 Leu Val Glu Ala Ala Thr Asp Ile Ala Lys Glu Leu Tyr Leu Gly Ala             100 105 110 Val Val Asp Arg Ser Ser Arg Arg Val Val Phe Met Ala Ser Thr Glu         115 120 125 Gly Gly Val Glu Ile Glu Lys Val Ala Glu Glu Thr Pro His Leu Ile     130 135 140 His Lys Val Ala Leu Asp Pro Leu Thr Gly Pro Met Pro Tyr Gln Gly 145 150 155 160 Arg Glu Leu Ala Phe Lys Leu Gly Leu Glu Gly Lys Leu Val Gln Gln                 165 170 175 Phe Thr Lys Ile Phe Met Gly Leu Ala Thr Ile Phe Leu Glu Arg Asp             180 185 190 Leu Ala Leu Ile Glu Ile Asn Pro Leu Val Ile Thr Lys Gln Gly Asp         195 200 205 Leu Ile Cys Leu Asp Gly Lys Leu Gly Ala Asp Gly Asn Ala Leu Phe     210 215 220 Arg Gln Pro Asp Leu Arg Glu Met Arg Asp Gln Ser Gln Glu Asp Pro 225 230 235 240 Arg Glu Ala Gln Ala Gln Trp Glu Leu Asn Tyr Val Ala Leu Asp                 245 250 255 Gly Asn Ile Gly Cys Met Val Asn Gly Ala Gly Leu Ala Met Gly Thr             260 265 270 Met Asp Ile Val Lys Leu His Gly Gly Glu Pro Ala Asn Phe Leu Asp         275 280 285 Val Gly Gly Gly Ala Thr Lys Glu Arg Val Thr Glu Ala Phe Lys Ile     290 295 300 Ile Leu Ser Asp Asp Lys Val Lys Ala Val Leu Val Asn Ile Phe Gly 305 310 315 320 Gly Ile Val Arg Cys Asp Leu Ile Ala Asp Gly Ile Ile Gly Ala Val                 325 330 335 Ala Glu Val Gly Val Val Val Val Val Val Val Arg Leu Glu Gly Asn             340 345 350 Asn Ala Glu Leu Gly Ala Lys Lys Leu Ala Asp Ser Gly Leu Asn Ile         355 360 365 Ile Ala Ala Lys Gly Leu Thr Asp Ala Ala Gln Gln Val Ala Ala     370 375 380 Val Glu Gly Lys Met Ser Ile Leu Ile Asp Lys Asn Thr Lys Val Ile 385 390 395 400 Cys Gln Gly Phe Thr Gly Ser Gln Gly Thr Phe His Ser Glu Gln Ala                 405 410 415 Ile Ala Tyr Gly Thr Lys Met Val Gly Gly Val Thr Pro Gly Lys Gly             420 425 430 Gly Thr Thr His Leu Gly Leu Pro Val Phe Asn Thr Val Arg Glu Ala         435 440 445 Val Ala Ala Thr Gly Ala Thr Ala Ser Val Ile Tyr Val Ala Pro     450 455 460 Phe Cys Lys Asp Ser Ile Leu Glu Ala Ile Asp Ala Gly Ile Lys Leu 465 470 475 480 Ile Ile Thr Ile Thr Glu Ile Pro Thr Leu Asp Met Leu Thr Val                 485 490 495 Lys Val Lys Leu Asp Glu Ala Gly Val Arg Met Ile Gly Pro Asn Cys             500 505 510 Pro Gly Val Ile Thr Pro Gly Gly Cys Lys Ile Gly Ile Gln Pro Gly         515 520 525 His Ile His Lys Pro Gly Lys Val Gly Ile Val Ser Ser Ser Gly Thr     530 535 540 Leu Thr Tyr Glu Ala Val Lys Gln Thr Thr Asp Tyr Gly Phe Gly Gln 545 550 555 560 Ser Thr Cys Val Gly Ile Gly Gly Asp Pro Ile Pro Gly Ser Asn Phe                 565 570 575 Ile Asp Ile Leu Glu Met Phe Glu Lys Asp Pro Gln Thr Glu Ala Ile             580 585 590 Val Met Ile Gly Glu Ile Gly         595 600 605 Tyr Ile Lys Glu His Val Thr Lys Pro Val Val Gly Tyr Ile Ala Gly     610 615 620 Val Thr Ala Pro Lys Gly Lys Arg Met Gly His Ala Gly Ala Ile Ile 625 630 635 640 Ala Gly Gly Lys Gly Thr Ala Asp Glu Lys Phe Ala Ala Leu Glu Ala                 645 650 655 Ala Gly Val Lys Thr Val Ser Ser Leu Ala Asp Ile Gly Glu Ala Leu             660 665 670 Lys Thr Val Leu Lys         675 <210> 8 <211> 2036 <212> DNA <213> E. coli <400> 8 atgaacttac atgaatatca ggcaaaacaa ctttttgccc gctatggctt accagcaccg 60 gtgggttatg cctgtactac tccgcgcgaa gcagaagaag ccgcttcaaa aatcggtgcc 120 ggtccgtggg tagtgaaatg tcaggttcac gctggtggcc gcggtaaagc gggcggtgtg 180 aaagttgtaa acagcaaaga agacatccgt gcttttgcag aaaactggct gggcaagcgt 240 ctggtaacgt atcaaacaga tgccaatggc caaccggtta accagattct ggttgaagca 300 gcgaccgata tcgctaaaga gctgtatctc ggtgccgttg ttgaccgtag ttcccgtcgt 360 gtggtcttta tggcctccac cgaaggcggc gtggaaatcg aaaaagtggc ggaagaaact 420 ccgcacctga tccataaagt tgcgcttgat ccgctgactg gcccgatgcc gtatcaggga 480 cgcgagctgg cgttcaaact gggtctggaa ggtaaactgg ttcagcagtt caccaaaatc 540 ttcatgggcc tggcgaccat tttcctggag cgcgacctgg cgttgatcga aatcaacccg 600 ctggtcatca ccaaacaggg cgatctgatt tgcctcgacg gcaaactggg cgctgacggc 660 aacgcactgt tccgccagcc tgatctgcgc gaaatgcgtg accagtcgca ggaagatccg 720 cgtgaagcac aggctgcaca gtgggaactg aactacgttg cgctggacgg taacatcggt 780 tgtatggtta acggcgcagg tctggcgatg ggtacgatgg acatcgttaa actgcacggc 840 ggcgaaccgg ctaacttcct tgacgttggc ggcggcgcaa ccaaagaacg tgtaaccgaa 900 gcgttcaaaa tcatcctctc tgacgacaaa gtgaaagccg ttctggttaa catcttcggc 960 ggtatcgttc gttgcgacct gatcgctgac ggtatcatcg gcgcggtagc agaagtgggt 1020 gttaacgtac cggtcgtggt acgtctggaa ggtaacaacg ccgaactcgg cgcgaagaaa 1080 ctggctgaca gcggcctgaa tattattgca gcaaaaggtc tgacggatgc agctcagcag 1140 gttgttgccg cagtggaggg gaaataatgt ccattttaat cgataaaaac accaaggtta 1200 tctgccaggg ctttaccggt agccagggga ctttccactc agaacaggcc attgcatacg 1260 gcactaaaat ggttggcggc gtaaccccag gtaaaggcgg caccacccac ctcggcctgc 1320 cggtgttcaa caccgtgcgt gaagccgttg ctgccactgg cgctaccgct tctgttatct 1380 acgtaccagc accgttctgc aaagactcca ttctggaagc catcgacgca ggcatcaaac 1440 tgattatcac catcactgaa ggcatcccga cgctggatat gctgaccgtg aaagtgaagc 1500 tggatgaagc aggcgttcgt atgatcggcc cgaactgccc aggcgttatc actccgggtg 1560 aatgcaaaat cggtatccag cctggtcaca ttcacaaacc gggtaaagtg ggtatcgttt 1620 cccgttccgg tacactgacc tatgaagcgg ttaaacagac cacggattac ggtttcggtc 1680 agtcgacctg tgtcggtatc ggcggtgacc cgatcccggg ctctaacttt atcgacattc 1740 tcgaaatgtt cgaaaaagat ccgcagaccg aagcgatcgt gatgatcggt gagatcggcg 1800 gtagcgctga agaagaagca gctgcgtaca tcaaagagca cgttaccaag ccagttgtgg 1860 gttacatcgc tggtgtgact gcgccgaaag gcaaacgtat gggccacgcg ggtgccatca 1920 ttgccggtgg gaaagggact gcggatgaga aattcgctgc tctggaagcc gcaggcgtga 1980 aaaccgttcg cagcctggcg gatatcggtg aagcactgaa aactgttctg aaataa 2036 <210> 9 <211> 538 <212> PRT <213> Clostridium kluyveri <400> 9 Met Ser Lys Gly Ile Lys Asn Ser Gln Leu Lys Lys Lys Asn Val Lys   1 5 10 15 Ala Ser Asn Val Ala Glu Lys Ile Glu Glu Lys Val Glu Lys Thr Asp              20 25 30 Lys Val Val Glu Lys Ala Ala Glu Val Thr Glu Lys Arg Ile Arg Asn          35 40 45 Leu Lys Leu Gln Glu Lys Val Val Thr Ala Asp Val Ala Ala Asp Met      50 55 60 Ile Glu Asn Gly Met Ile Val Ala Ile Ser Gly Phe Thr Pro Ser Gly  65 70 75 80 Tyr Pro Lys Glu Val Pro Lys Ala Leu Thr Lys Lys Val Asn Ala Leu                  85 90 95 Glu Glu Glu Phe Lys Val Thr Leu Tyr Thr Gly Ser Ser Thr Gly Ala             100 105 110 Asp Ile Asp Gly Glu Trp Ala Lys Ala Gly Ile Ile Glu Arg Arg Ile         115 120 125 Pro Tyr Gln Thr Asn Ser Asp Met Arg Lys Lys Ile Asn Asp Gly Ser     130 135 140 Ile Lys Tyr Ala Asp Met His Leu Ser His Met Ala Gln Tyr Ile Asn 145 150 155 160 Tyr Ser Val Ile Pro Lys Val Asp Ile Ala Ile Ile Glu Ala Val Ala                 165 170 175 Ile Thr Glu Glu Gly Asp Ile Ile Pro Ser Thr Gly Ile Gly Asn Thr             180 185 190 Ala Thr Phe Val Glu Asn Ale Asp Lys Val Ile Val Glu Ile Asn Glu         195 200 205 Ala Gln Pro Leu Glu Leu Glu Gly Met Ala Asp Ile Tyr Thr Leu Lys     210 215 220 Asn Pro Pro Arg Arg Glu Pro Ile Pro Ile Val Asn Ala Gly Asn Arg 225 230 235 240 Ile Gly Thr Thr Tyr Val Thr Cys Gly Ser Glu Lys Ile Cys Ala Ile                 245 250 255 Val Met Thr Asn Thr Gln Asp Lys Thr Arg Pro Leu Thr Glu Val Ser             260 265 270 Pro Val Ser Gln Ala Ile Ser Asp Asn Leu Ile Gly Phe Leu Asn Lys         275 280 285 Glu Val Glu Glu Gly Lys Leu Pro Lys Asn Leu Leu Pro Ile Gln Ser     290 295 300 Gly Val Gly Ser Val Ala Asn Ala Val Leu Ala Gly Leu Cys Glu Ser 305 310 315 320 Asn Phe Lys Asn Leu Ser Cys Tyr Thr Glu Val Ile Gln Asp Ser Met                 325 330 335 Leu Lys Leu Ile Lys Cys Gly Lys Ala Asp Val Val Ser Gly Thr Ser             340 345 350 Ile Ser Pro Ser Pro Glu Met Leu Pro Glu Phe Ile Lys Asp Ile Asn         355 360 365 Phe Phe Arg Glu Lys Ile Val Leu Arg Pro Gln Glu Ile Ser Asn Asn     370 375 380 Pro Glu Ile Ala Arg Arg Ile Gly Val Ile Ser Ile Asn Thr Ala Leu 385 390 395 400 Glu Val Asp Ile Tyr Gly Asn Val Asn Ser Thr His Val Met Gly Ser                 405 410 415 Lys Met Met Asn Gly Ile Gly Gly Ser Gly Asp Phe Ala Arg Asn Ala             420 425 430 Tyr Leu Thr Ile Phe Thr Thr Glu Ser Ile Ala Lys Lys Gly Asp Ile         435 440 445 Ser Ser Ile Val Pro Met Val Ser Ser Val Hisp His Thr Glu His Asp     450 455 460 Val Met Val Ile Val Thr Glu Gln Gly Val Ala Asp Leu Arg Gly Leu 465 470 475 480 Ser Pro Arg Glu Lys Ala Val Ala Ile Ile Glu Asn Cys Val His Pro                 485 490 495 Asp Tyr Lys Asp Met Leu Met Glu Tyr Phe Glu Glu Ala Cys Lys Ser             500 505 510 Ser Gly Gly Asn Thr Pro His Asn Leu Glu Lys Ala Leu Ser Trp His         515 520 525 Thr Lys Phe Ile Lys Thr Gly Ser Met Lys     530 535 <210> 10 <211> 1617 <212> DNA <213> Clostridium kluyveri <400> 10 atgagtaaag ggattaagaa ctcgcaacta aaaaaaaaaa atgtgaaggc cagtaatgtg 60 gcagaaaaga ttgaagagaa agttgaaaaa acggataagg ttgttgaaaa agccgctgag 120 gttacagaga aacggattag aaacctgaag ctgcaggaga aagttgttac agcggatgtg 180 gcggctgata tgattgaaaa tggcatgatt gtggcaatca gcggttttac tccgtccggt 240 tatccaaagg aagtccctaa agcactgact aaaaaagtta atgccctgga ggaggagttc 300 aaggtcacct tatataccgg gtcaagcacg ggggccgaca tcgacgggga atgggcaaag 360 gcaggaatca tagaacggcg tatcccctac cagacaaatt ctgacatgcg aaaaaaaata 420 aatgacggtt ctattaagta cgctgatatg catttaagcc atatggctca atatattaat 480 tattctgtca ttcctaaagt cgatatagct ataatagaag cggtagctat tacggaagaa 540 ggggatataa ttccttcgac gggaattggc aataccgcga cttttgtgga aaacgcggac 600 aaagtgatag tggaaattaa cgaagcccaa ccgctggaat tggagggcat ggcagacata 660 tacacattaa aaaacccccc gcgtagagag ccgattccaa tagttaatgc tggcaatcgc 720 atagggacca catatgtgac ctgtggctcg gaaaaaatct gcgccatcgt catgacaaat 780 acgcaagaca aaacaagacc tcttacagag gtgtctcctg tatctcaggc catctccgac 840 aatctgatag gttttttaaa caaagaagtg gaagagggca aattacctaa aaacctgctc 900 cccatacagt caggagttgg tagtgtcgca aatgcggttt tggccggtct ttgtgaatca 960 aactttaaaa acctaagttg ttacacggag gttatccagg atagcatgct gaagcttata 1020 aaatgtggaa aagcagatgt ggtgtcaggc acctccataa gtccatcacc ggagatgctg 1080 cctgagttca tcaaggacat aaacttcttt agagaaaaga tagtattaag accacaggaa 1140 atcagcaata acccagagat agcacgcaga atcggtgtga tatccataaa caccgccttg 1200 gaagtagaca tatatggtaa tgtaaacagt acgcacgtta tgggaagcaa aatgatgaat 1260 ggcataggcg gttctggcga ctttgcccgc aatgcatatc tcactatctt cactacagag 1320 tctatcgcca aaaaaggcga tatctcaagc atagtgccta tggtatccca tgtggatcat 1380 accgaacatg atgtaatggt catcgttacc gaacagggag tagcggatct gcgcggtctt 1440 tctcctaggg aaaaggcggt ggctataatc gaaaattgcg ttcatccgga ctataaggat 1500 atgctgatgg agtattttga agaagcgtgc aaatcgtcag gtgggaacac cccacacaat 1560 cttgaaaaag ctctttcatg gcacacaaaa tttataaaaa cgggtagcat gaaataa 1617 <210> 11 <211> 451 <212> PRT <213> Porphyromonas gingivalis <400> 11 Met Glu Ile Lys Glu Met Val Ser Leu Ala Arg Lys Ala Gln Lys Glu   1 5 10 15 Tyr Gln Ala Thr His Asn Gln Glu Ala Val Asp Asn Ile Cys Arg Ala              20 25 30 Ala Ala Lys Val Ile Tyr Glu Asn Ala Ala Ile Leu Ala Arg Glu Ala          35 40 45 Val Asp Glu Thr Gly Met Gly Val Tyr Glu His Lys Val Ala Lys Asn      50 55 60 Gln Gly Lys Ser Lys Gly Val Trp Tyr Asn Leu His Asn Lys Lys Ser  65 70 75 80 Ile Gly Ile Leu Asn Ile Asp Glu Arg Thr Gly Met Ile Glu Ile Ala                  85 90 95 Lys Pro Ile Gly Val Val Gly Ala Val Thr Pro Thr Thr Asn Pro Ile             100 105 110 Val Thr Pro Met Ser Asn Ile Ile Phe Ala Leu Lys Thr Cys Asn Ala         115 120 125 Ile Ile Ile Ala Pro His Pro Arg Ser Lys Lys Cys Ser Ala His Ala     130 135 140 Val Arg Leu Ile Lys Glu Ala Ile Ala Pro Phe Asn Val Pro Glu Gly 145 150 155 160 Met Val Gln Ile Ile Glu Glu Pro Ser Ile Glu Lys Thr Gln Glu Leu                 165 170 175 Met Gly Ala Val Asp Val Val Ala Thr Gly Gly Met Gly Met Val             180 185 190 Lys Ser Ala Tyr Ser Ser Gly Lys Ser Ser Phe Gly Val Gly Ala Gly         195 200 205 Asn Val Gln Val Ile Val Asp Ser Asn Ile Asp Phe Glu Ala Ala Ala     210 215 220 Glu Lys Ile Ile Thr Gly Arg Ala Phe Asp Asn Gly Ile Ile Cys Ser 225 230 235 240 Gly Glu Gln Ser Ile Ile Tyr Asn Glu Ala Asp Lys Glu Ala Val Phe                 245 250 255 Thr Ala Phe Arg Asn His Gly Ala Tyr Phe Cys Asp Glu Ala Glu Gly             260 265 270 Asp Arg Ala Arg Ala Ala Ile Phe Glu Asn Gly Ala Ile Ala Lys Asp         275 280 285 Val Val Gly Gln Ser Val Ala Phe Ile Ala Lys Lys Ala Asn Ile Asn     290 295 300 Ile Pro Glu Gly Thr Arg Ile Le Val Val Glu Ala Arg Gly Val Gly 305 310 315 320 Ala Glu Asp Val Ile Cys Lys Glu Lys Met Cys Pro Val Met Cys Ala                 325 330 335 Leu Ser Tyr Lys His Phe Glu Glu Gly Val Glu Ile Ala Arg Thr Asn             340 345 350 Leu Ala Asn Glu Gly Asn Gly His Thr Cys Ala Ile His Ser Asn Asn         355 360 365 Gln Ala His Ile Ile Leu Ala Gly Ser Glu Leu Thr Val Ser Arg Ile     370 375 380 Val Val Asn Ala Pro Ser Ala Thr Thr Ala Gly Gly His Ile Gln Asn 385 390 395 400 Gly Leu Ala Val Thr Asn Thr Leu Gly Cys Gly Ser Trp Gly Asn Asn                 405 410 415 Ser Ile Ser Glu Asn Phe Thr Tyr Lys His Leu Leu Asn Ile Ser Arg             420 425 430 Ile Ala Pro Leu Asn Ser Ser Ile His Ile Pro Asp Asp Lys Glu Ile         435 440 445 Trp Glu Leu     450 <210> 12 <211> 1356 <212> DNA <213> Porphyromonas gingivalis <400> 12 atggaaataa aagagatggt gtcgttggca aggaaagctc agaaggaata tcaagcgacc 60 cataatcaag aagcagttga taacatttgc cgagctgcag caaaagtgat ttatgaaaat 120 gcagctatac tggctcgcga agcagtagac gaaaccggca tgggcgtata tgaacataaa 180 gtggccaaga atcaggggaa atccaaaggc gtctggtaca atttgcacaa taaaaaatcg 240 atcggtatct taaatataga cgagagaacc gggatgatcg agatagcaaa acctatcggg 300 gttgttggag ccgtaacccc gacgacaaac ccgattgtga ctccaatgag caacatcatt 360 tttgccctta agacatgcaa tgccattatt atcgccccac atcccagatc caaaaaatgc 420 tcagcacatg cagttcgtct gataaaggaa gcaatcgctc cgtttaatgt cccggaggga 480 atggttcaga tcattgaaga gcccagcatc gagaaaactc aggaactaat gggcgccgtg 540 gatgtggtag ttgcgacggg tggtatgggt atggtgaaat ctgcatattc ttcagggaag 600 ccttcttttg gtgtaggagc cggtaacgtt caagtgatcg tggatagtaa tatcgatttt 660 gaagctgcgg cagaaaaaat tatcaccggc cgtgctttcg acaatgggat catctgttca 720 ggcgaacaga gtatcatcta caacgaagct gacaaggaag ctgtcttcac agccttccgc 780 aaccatggtg catatttttg tgatgaagcg gagggagatc gggcccgtgc tgcgattttt 840 gagaatggcg ccatcgcgaa agatgtagtc ggccagagcg ttgcctttat cgcgaagaaa 900 gcaaatatca atataccgga gggtacccgt attctggttg ttgaagctcg cggcgtcgga 960 gcagaggatg tcatatgtaa ggaaaaaatg tgtccagtta tgtgcgcctt aagctacaag 1020 cacttcgagg aaggtgtaga aatcgcacgt acgaacttgg ccaacgaagg taacggccat 1080 acctgtgcga tccattccaa caatcaggcg catatcatac tggcaggttc agaactgacg 1140 gtttcgcgga tcgtggtcaa tgcgccgagt gccactacag caggcggtca catccaaaat 1200 ggtctggcag tgacaaatac gctcggatgc gggagttggg gtaataactc tatctccgag 1260 aactttactt ataaacacct gttaaacatt agccgcatag cgccgcttaa ttcaagcatt 1320 cacattcctg atgacaaaga gatctgggaa ctctaa 1356 <210> 13 <211> 371 <212> PRT <213> Porphyromonas gingivalis <400> 13 Met Gln Leu Phe Lys Leu Lys Ser Val Thr His His Phe Asp Thr Phe   1 5 10 15 Ala Glu Phe Ala Lys Glu Phe Cys Leu Gly Glu Arg Asp Leu Val Ile              20 25 30 Thr Asn Glu Phe Ile Tyr Glu Pro Tyr Met Lys Ala Cys Gln Leu Pro          35 40 45 Cys His Phe Val Met Gln Glu Lys Tyr Gly Gln Gly Glu Pro Ser Asp      50 55 60 Glu Met Met Asn Asn Ile Leu Ala Asp Ile Arg Asn Ile Gln Phe Asp  65 70 75 80 Arg Val Ile Gly Ile Gly Gly Gly Thr Val Ile Asp Ile Ser Lys Leu                  85 90 95 Phe Val Leu Lys Gly Leu Asn Asp Val Leu Asp Ala Phe Asp Arg Lys             100 105 110 Ile Pro Leu Ile Lys Glu Lys Glu Leu Ile Ile Val Pro Thr Thr Cys         115 120 125 Gly Thr Gly Ser Glu Val Thr Asn Ile Ser Ile Ala Glu Ile Lys Ser     130 135 140 Arg His Thr Lys Met Gly Leu Ala Asp Asp Ala Ile Val Ala Asp His 145 150 155 160 Ala Ile Ile Ile Pro Glu Leu Leu Lys Ser Leu Pro Phe His Phe Tyr                 165 170 175 Ala Cys Ser Ala Ile Asp Ala Leu Ile His Ala Ile Glu Ser Tyr Val             180 185 190 Ser Pro Lys Ala Ser Pro Tyr Ser Arg Leu Phe Ser Glu Ala Ala Trp         195 200 205 Asp Ile Leu Glu Val Phe Lys Lys Ile Ala Glu His Gly Pro Glu     210 215 220 Tyr Arg Phe Glu Lys Leu Gly Glu Met Ile Met Ala Ser Asn Tyr Ala 225 230 235 240 Gly Ile Ala Phe Gly Asn Ala Gly                 245 250 255 Tyr Pro Leu Gly Gly Asn Tyr His Val Gly Gly Glu Ala Asn Tyr             260 265 270 Gln Phe Phe Thr Glu Val Phe Lys Val Tyr Gln Lys Lys Asn Pro Phe         275 280 285 Gly Tyr Ile Val Glu Leu Asn Trp Lys Leu Ser Lys Ile Leu Asn Cys     290 295 300 Gln Pro Glu Tyr Val Tyr Pro Lys Leu Asp Glu Leu Leu Gly Cys Leu 305 310 315 320 Leu Thr Lys Lys Pro Leu His Glu Tyr Gly Met Lys Asp Glu Glu Val                 325 330 335 Arg Gly Phe Ala Glu Ser Val Leu Lys Thr Gln Gln Arg Leu Leu Ala             340 345 350 Asn Asn Tyr Val Glu Leu Thr Val Asp Glu Ile Glu Gly Ile Tyr Arg         355 360 365 Arg Leu Tyr     370 <210> 14 <211> 1116 <212> DNA <213> Porphyromonas gingivalis <400> 14 atgcaactgt tcaaactgaa atcagtcaca catcacttcg atactttcgc ggaatttgcc 60 aaagagttct gtcttggaga acgtgattta gtaattacca acgaattcat ttacgaaccg 120 tatatgaagg catgtcagtt gccctgccat tttgttatgc aggagaaata tgggcaaggc 180 gagccatctg acgagatgat gaataacatc ttggcagaca tccgtaatat ccagtttgac 240 cgcgtgatcg gtattggggg tggtacggtt attgacatct cgaaattatt tgtgctgaaa 300 ggactaaatg atgtgctcga tgcgttcgat cgcaagatac cgctgattaa agagaaagaa 360 ctgatcattg tgcccaccac atgcgggacg ggtagcgagg tgacgaatat ttcgatcgcg 420 gagatcaaaa gccgtcatac caaaatgggt ttggctgacg atgctattgt tgcagaccac 480 gcgatcatca taccagagct tctgaaaagc ctgccgttcc atttttatgc atgcagtgca 540 atagatgctc tgatccatgc catcgagtca tatgtttctc ctaaagccag tccatattct 600 cgtctgttca gtgaggcggc atgggatatt atcctggagg tattcaagaa aatagccgaa 660 cacggccctg aataccgctt tgagaagctg ggagaaatga tcatggcctc caactatgct 720 ggtatagcct tcgggaatgc aggcgtgggt gccgttcacg ctctaagcta tccattggga 780 ggcaattatc atgtgccgca tggcgaggct aactatcagt tttttacaga ggtctttaaa 840 gtataccaaa agaaaaatcc tttcggctat atagtcgaac tcaactggaa gctgtccaag 900 attctgaact gtcagcctga atacgtctat ccgaaactgg atgagttact cggctgtctt 960 cggaccaaaa aaccgctgca cgaatacggc atgaaagatg aagaggtacg tggatttgcg 1020 gaatcagtgc ttaagactca gcagcggttg ctcgcgaata attatgttga gcttactgtt 1080 gatgaaattg aaggtatcta cagacgactg tactaa 1116 <210> 15 <211> 431 <212> PRT <213> Porphyromonas gingivalis <400> 15 Met Lys Asp Val Leu Ala Glu Tyr Ala Ser Arg Ile Val Ser Ala Glu   1 5 10 15 Glu Ala Val Lys His Ile Lys Asn Gly Glu Arg Val Ala Leu Ser His              20 25 30 Ala Ala Gly Val Pro Gln Ser Cys Val Asp Ala Leu Val Gln Gln Ala          35 40 45 Asp Leu Phe Gln Asn Val Glu Ile Tyr His Met Leu Cys Leu Gly Glu      50 55 60 Gly Lys Tyr Met Ala Pro Glu Met Ala Pro His Phe Arg His Ile Thr  65 70 75 80 Asn Phe Val Gly Gly Asn Ser Arg Lys Ala Val Glu Glu Asn Arg Ala                  85 90 95 Asp Phe Ile Pro Val Phe Phe Tyr Glu Val Pro Ser Met Ile Arg Lys             100 105 110 Asp Ile Leu His Ile Asp Val Ala Ile Val Gln Leu Ser Met Pro Asp         115 120 125 Glu Asn Gly Tyr Cys Ser Phe Gly Val Ser Cys Asp Tyr Ser Lys Pro     130 135 140 Ala Ala Glu Ser Ala His Leu Val Ile Gly Glu Ile Asn Arg Gln Met 145 150 155 160 Pro Tyr Val His Gly Asp Asn Leu Ile His Ile Ser Lys Leu Asp Tyr                 165 170 175 Ile Val Met Ala Asp Tyr Pro Ile Tyr Ser Leu Ala Lys Pro Lys Ile             180 185 190 Gly Glu Val Glu Glu Ala Ile Gly Arg Asn Cys Ala Glu Leu Ile Glu         195 200 205 Asp Gly Ala Thr Leu Gly Leu Gly Ile Gly Ala Ile Pro Asp Ala Ala     210 215 220 Leu Leu Phe Leu Lys Asp Lys Lys Asp Leu Gly Ile His Thr Glu Met 225 230 235 240 Phe Ser Asp Gly Val Val Glu Leu Val Arg Ser Gly Val Ile Thr Gly                 245 250 255 Lys Lys Lys Thr Leu His Pro Gly Lys Met Val Ala Thr Phe Leu Met             260 265 270 Gly Ser Glu Asp Val Tyr His Phe Ile Asp Lys Asn Pro Asp Val Glu         275 280 285 Leu Tyr Pro Val Asp Tyr Val Asn Asp Pro Arg Val Ile Ala Gln Asn     290 295 300 Asp Asn Met Val Ser Ile Asn Ser Cys Ile Glu Ile Asp Leu Met Gly 305 310 315 320 Gln Val Val Ser Glu Cys Ile Gly Ser Lys Gln Phe Ser Gly Thr Gly                 325 330 335 Gly Gln Val Asp Tyr Val Arg Gly Ala Ala Trp Ser Lys Asn Gly Lys             340 345 350 Ser Ile Met Ala Ile Pro Ser Thr Ala Lys Asn Gly Thr Ala Ser Arg         355 360 365 Ile Val Pro Ile Ile Ala Glu Gly Ala Ala Val Thr Thr Leu Arg Asn     370 375 380 Glu Val Asp Tyr Val Val Thr Glu Tyr Gly Ile Ala Gln Leu Lys Gly 385 390 395 400 Lys Ser Leu Arg Gln Arg Ala Glu Ala Leu Ile Ala Ile Ala His Pro                 405 410 415 Asp Phe Arg Glu Glu Leu Thr Lys His Leu Arg Lys Arg Phe Gly             420 425 430 <210> 16 <211> 1296 <212> DNA <213> Porphyromonas gingivalis <400> 16 atgaaagacg tgttagcgga atatgcctcc cgaattgttt cggccgaaga ggcagtcaaa 60 catatcaaaa atggagagcg tgtcgcttta tcacatgctg ccggagttcc tcagagttgt 120 gttgacgcac tggtgcaaca ggcggacctg tttcagaatg tggagattta ccacatgctg 180 tgtctcggcg aaggaaaata tatggcacct gaaatggccc ctcacttccg gcacataacc 240 aattttgttg gtggtaactc tcgtaaagca gtggaggaaa atagagccga cttcattccg 300 gtattctttt atgaagtgcc atcaatgatt cggaaagata tccttcatat agatgtggcc 360 attgtccaac tctcaatgcc agatgagaat ggttactgca gctttggcgt atcttgcgat 420 tatagcaaac cggcggcgga atcggcgcat ttagttattg gggaaatcaa ccgtcagatg 480 ccatatgtgc atggtgacaa cttgattcac atatcgaagt tggattacat cgtgatggcg 540 gattacccaa tttattctct ggcgaagccc aaaatcggag aagtagagga agctatcggc 600 cgtaactgtg ccgagcttat tgaagatggt gccaccctac agctgggtat cggcgcgatt 660 ccggatgcag ctctgctgtt tctgaaggac aaaaaagatc tggggattca tactgaaatg 720 ttctccgatg gcgttgttga actggtgcgc agtggtgtaa ttactggaaa aaaaaagaca 780 ttgcatcccg gtaagatggt cgcgacgttt cttatgggat cagaagacgt gtatcatttc 840 atcgacaaga atccggatgt ggaactgtat ccggttgatt acgtcaatga tccgagggtt 900 atcgctcaga atgataatat ggtcagcatc aatagctgta tcgagatcga tctaatgggc 960 caagtggtga gcgagtgcat aggctccaaa cagtttagtg gcaccggggg tcaagtagat 1020 tatgtccgcg gggcagcttg gtctaaaaac ggcaaaagca tcatggcaat tccctcaaca 1080 gccaaaaacg gtactgcatc tcggatagtt cctataattg cagagggcgc tgctgtaaca 1140 accctccgca acgaagtcga ctacgttgtt acggaatatg ggatagcaca gttaaaaggt 1200 aagagtttgc gtcagcgcgc agaagctctt attgcgatag cccacccgga ctttagagag 1260 gaactgacga agcatctgcg caaacgtttt ggttaa 1296 <210> 17 <211> 858 <212> PRT <213> Clostridium acetobutylicum <400> 17 Met Lys Val Thr Asn Gln Lys Glu Leu Lys Gln Lys Leu Asn Glu Leu   1 5 10 15 Arg Glu Ala Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val Asp              20 25 30 Lys Ile Phe Lys Gln Cys Ala Ile Ala Ala Ala Lys Glu Arg Ile Asn          35 40 45 Leu Ala Lys Leu Ala Val Glu Glu Thr Gly Ile Gly Leu Val Glu Asp      50 55 60 Lys Ile Ile Lys Asn His Phe Ala Ala Glu Tyr Ile Tyr Asn Lys Tyr  65 70 75 80 Lys Asn Glu Lys Thr Cys Gly Ile Ile Asp His Asp Asp Ser Leu Gly                  85 90 95 Ile Thr Lys Val Ala Glu Pro Ile Gly Ile Val Ala Ala Ile Val Pro             100 105 110 Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu         115 120 125 Lys Thr Arg Asn Ala Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys     130 135 140 Ser Thr Ile Ala Ala Lys Leu Ile Leu Asp Ala Ala Val Lys Ala 145 150 155 160 Gly Ala Pro Lys Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu                 165 170 175 Leu Ser Gln Asp Leu Met Ser Glu Ala Asp Ile Ile Leu Ala Thr Gly             180 185 190 Gly Pro Ser Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile         195 200 205 Gly Val Gly Ala Gly Asn Thr Pro Ala Ile Ile Asp Glu Ser Ala Asp     210 215 220 Ile Asp Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn 225 230 235 240 Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Leu Val Met Asn Ser Ile                 245 250 255 Tyr Glu Lys Val Lys Glu Glu Phe Val Lys Arg Gly Ser Tyr Ile Leu             260 265 270 Asn Gln Asn Glu Ile Ala Lys Ile Lys Glu Thr Met Phe Lys Asn Gly         275 280 285 Ala Ile Asl Ala Asp Ile Val Gly Lys Ser Ala Tyr Ile Ile Ala Lys     290 295 300 Met Ala Gly Ile Glu Val Pro Gln Thr Thr Lys Ile Leu Ile Gly Glu 305 310 315 320 Val Gln Ser Val Glu Lys Ser Glu Leu Phe Ser His Glu Lys Leu Ser                 325 330 335 Pro Val Leu Ala Met Tyr Lys Val Lys Asp Phe Asp Glu Ala Leu Lys             340 345 350 Lys Ala Gln Arg Leu Ile Glu Leu Gly Gly Ser Gly His Thr Ser Ser         355 360 365 Leu Tyr Ile Asp Ser Gln Asn Asn Lys Asp Lys Val Lys Glu Phe Gly     370 375 380 Leu Ala Met Lys Thr Ser Arg Thr Phe Ile Asn Met Pro Ser Ser Gln 385 390 395 400 Gly Ala Ser Gly Asp Leu Tyr Asn Phe Ala Ile Ala Pro Ser Phe Thr                 405 410 415 Leu Gly Cys Gly Thr Trp Gly Gly Asn Ser Val Ser Gln Asn Val Glu             420 425 430 Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn         435 440 445 Met Leu Trp Phe Lys Val Pro Gln Lys Ile Tyr Phe Lys Tyr Gly Cys     450 455 460 Leu Arg Phe Ala Leu Lys Glu Leu Lys Asp Met Asn Lys Lys Arg Ala 465 470 475 480 Phe Ile Val Thr Asp Lys Asp Leu Phe Lys Leu Gly Tyr Val Asn Lys                 485 490 495 Ile Thr Lys Val Leu Asp Glu Ile Asp Ile Lys Tyr Ser Ile Phe Thr             500 505 510 Asp Ile Lys Ser Asp Pro Thr Ile Asp Ser Val Lys Lys Gly Ala Lys         515 520 525 Glu Met Leu Asn Phe Glu Pro Asp Thr Ile Ile Ser Ile Gly Gly Gly     530 535 540 Ser Pro Met Asp Ala Ala Lys Val Met His Leu Leu Tyr Glu Tyr Pro 545 550 555 560 Glu Ala Glu Ile Glu Asn Leu Ala Ile Asn Phe Met Asp Ile Arg Lys                 565 570 575 Arg Ile Cys Asn Phe Pro Lys Leu Gly Thr Lys Ala Ile Ser Val Ala             580 585 590 Ile Pro Thr Thr Ala Gly Thr Gly Ser Glu Ala Thr Pro Phe Ala Val         595 600 605 Ile Thr Asn Asp Glu Thr Gly Met Lys Tyr Pro Leu Thr Ser Tyr Glu     610 615 620 Leu Thr Pro As Met Ale Ile Asle Asp Thr Glu Leu Met Leu Asn Met 625 630 635 640 Pro Arg Lys Leu Thr Ala Ala Thr Gly Ile Asp Ala Leu Val His Ala                 645 650 655 Ile Glu Ala Tyr Val Ser Val Ala Thr Asp Tyr Thr Asp Glu Leu             660 665 670 Ala Leu Arg Ala Ile Lys Met Ile Phe Lys Tyr Leu Pro Arg Ala Tyr         675 680 685 Lys Asn Gly Thr Asn Asp Ile Glu Ala Arg Glu Lys Met Ala His Ala     690 695 700 Ser Asn Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly Val Cys 705 710 715 720 His Ser Met Ala His Lys Leu Gly Ala Met His His Val Pro His Gly                 725 730 735 Ile Ala Cys Ala Val Leu Ile Glu Glu Val Ile Lys Tyr Asn Ala Thr             740 745 750 Asp Cys Pro Thr Lys Gln Thr Ala Phe Pro Gln Tyr Lys Ser Pro Asn         755 760 765 Ala Lys Arg Lys Tyr Ala Glu Ile Ala Glu Tyr Leu Asn Leu Lys Gly     770 775 780 Thr Ser Asp Thr Glu Lys Val Thr Ala Leu Ile Glu Ala Ile Ser Lys 785 790 795 800 Leu Lys Ile Asp Leu Ser Ile Pro Gln Asn Ile Ser Ala Ala Gly Ile                 805 810 815 Asn Lys Lys Asp Phe Tyr Asn Thr Leu Asp Lys Met Ser Glu Leu Ala             820 825 830 Phe Asp Gln Cys Thr Thr Ala Asn Pro Arg Tyr Pro Leu Ile Ser         835 840 845 Glu Leu Lys Asp Ile Tyr Ile Lys Ser Phe     850 855 <210> 18 <211> 2577 <212> DNA <213> Clostridium acetobutylicum <400> 18 atgaaagtca ccaaccagaa agagctgaaa cagaaactga acgaactgcg tgaagcccag 60 aagaagttcg ctacgtacac ccaggaacag gtggacaaaa tcttcaaaca gtgcgcgatt 120 gctgctgcaa aagaacgtat caacctggct aaactggccg tggaagagac gggcattggt 180 ctggtggaag acaagatcat caaaaaccac tttgcggcgg aatacatcta caacaagtac 240 aaaaacgaga aaacttgcgg catcatcgac cacgatgatt ccctgggcat caccaaagtg 300 gcagaaccta tcggtattgt tgcagcaatt gtaccgacta ctaacccgac ttctaccgct 360 attttcaagt ctctgatttc tctgaaaacc cgcaacgcga ttttcttctc tccgcaccca 420 cgtgcgaaaa aatccaccat cgctgccgcc aaactgatcc tggacgcggc agttaaagcg 480 ggtgctccga aaaatatcat tggttggatc gatgaaccgt ctatcgaact gagccaggat 540 ctgatgtccg aagcagacat tatcctggca accggcggtc cgtctatggt aaaagccgcc 600 tactcttctg gcaaaccggc aattggtgtt ggtgctggta acacgccggc gattatcgac 660 gagtccgcag acatcgatat ggcagtttcc tctatcattc tgtccaaaac ctacgataac 720 ggcgtgatct gcgcgagcga acagtccatc ctggttatga attctatcta tgaaaaggtc 780 aaggaagaat ttgttaagcg tggcagctac atcctgaacc agaacgagat cgcgaaaatc 840 aaagaaacta tgttcaaaaa cggtgccatc aatgccgaca tcgtcggcaa atctgcttac 900 attattgcca aaatggctgg tatcgaagtg ccgcagacca cgaagatcct gatcggtgag 960 gtacagagcg ttgaaaagtc tgaactgttc tctcatgaaa aactgtcccc ggtcctggct 1020 atgtacaaag taaaagactt cgacgaagca ctgaaaaaag cgcaacgtct gatcgagctg 1080 ggtggtagcg gccacacctc tagcctgtac atcgacagcc agaacaacaa agataaagtt 1140 aaagaattcg gcctggcaat gaaaaccagc cgcaccttta ttaacatgcc ttctagccaa 1200 ggtgcttctg gcgacctgta taacttcgct attgcgcctt cctttaccct gggttgcggt 1260 acctggggcg gtaacagcgt ttcccaaaac gttgaaccga aacacctgct gaacattaaa 1320 tctgtagcag aacgccgtga gaacatgctg tggtttaaag ttccgcagaa aatctacttc 1380 aagtacggtt gtctgcgctt cgctctgaaa gaactgaagg atatgaacaa gaaacgtgcg 1440 ttcatcgtga ctgataaaga tctgttcaaa ctgggctacg ttaacaaaat cactaaagta 1500 ctggacgaaa tcgatattaa gtattccatc tttaccgaca tcaaatctga cccgaccatc 1560 gattccgtaa aaaagggtgc taaggaaatg ctgaacttcg aaccggacac tattatcagc 1620 atcggcggtg gctctccgat ggatgcagca aaagtgatgc atctgctgta cgaatacccg 1680 gaagcggaaa tcgaaaacct ggcgatcaat ttcatggaca tccgtaaacg tatctgcaat 1740 tttccgaagc tgggtacgaa agccatttcc gttgcgattc cgactaccgc gggtactggt 1800 tctgaagcga ccccgttcgc tgttattact aacgatgaaa ctggtatgaa atacccactg 1860 acgagctatg agctgacccc aaacatggca atcattgata ccgagctgat gctgaatatg 1920 ccgcgtaaac tgaccgcggc gactggcatc gacgccctgg ttcacgcgat cgaagcttat 1980 gtttctgtca tggccaccga ttatacggac gaactggctc tgcgtgctat caaaatgatt 2040 ttcaaatatc tgcctcgcgc gtacaagaac ggcaccaacg atattgaggc tcgtgaaaaa 2100 atggcacacg ccagcaacat cgcaggcatg gcattcgcta acgcttttct gggcgtatgc 2160 cattccatgg ctcataaact gggtgcaatg caccacgttc cacacggcat cgcgtgtgcg 2220 gtgctgatcg aagaggtgat caaatacaac gctactgact gtccgactaa acaaaccgcg 2280 tttccgcagt acaaatcccc aaatgcgaaa cgtaaatatg cggagatcgc cgaatatctg 2340 aacctgaaag gcacctccga caccgaaaaa gtgaccgctc tgattgaagc catcagcaaa 2400 ctgaaaattg acctgtctat cccgcagaac atcagcgcgg caggtatcaa caaaaaagat 2460 ttctataaca ccctggataa aatgagcgag ctggcgttcg atgaccagtg tacgaccgca 2520 aacccgcgct acccgctgat ctccgaactg aaagacattt atattaaatc cttctaa 2577 <210> 19 <211> 468 <212> PRT <213> Clostridium beijerinckii <400> 19 Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Val Lys   1 5 10 15 Thr Asn Gly Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser              20 25 30 Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Ser Ala Val          35 40 45 His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu      50 55 60 Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Gln Asn Lys Glu Val  65 70 75 80 Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp                  85 90 95 Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu             100 105 110 Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val         115 120 125 Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn     130 135 140 Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly 145 150 155 160 Asn Ala Val Val Phe Asn Gly His Pro Cys Ala Lys Lys Cys Val Ala                 165 170 175 Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ser Ser Cys Gly Gly Pro             180 185 190 Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp         195 200 205 Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly     210 215 220 Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly 225 230 235 240 Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile                 245 250 255 Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn             260 265 270 Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala         275 280 285 Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn     290 295 300 Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn 305 310 315 320 Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala                 325 330 335 Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys             340 345 350 Cys Ile Ile Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu         355 360 365 Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu     370 375 380 Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala 385 390 395 400 Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu                 405 410 415 Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val             420 425 430 Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr         435 440 445 Gly Glu Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys     450 455 460 Val Leu Ala Gly 465 <210> 20 <211> 1407 <212> DNA <213> Clostridium beijerinckii <400> 20 atgaataagg atacgttgat cccgaccacc aaggatctga aggtcaagac caatggcgag 60 aacatcaatc ttaaaaacta caaagacaac agctcctgtt tcggagtgtt tgaaaatgtg 120 gaaaacgcga tctcctcagc agtacacgcg caaaagatcc tctctttgca ctacactaag 180 gaacagcgtg aaaagattat cacggagatc cgcaaagcgg cactgcagaa caaagaggtc 240 ctggctacaa tgatcttgga ggagacacat atgggtcgct acgaggacaa gatcctcaag 300 cacgagcttg ttgctaagta cacccccggc accgaggatc ttaccaccac cgcctggtct 360 ggcgataatg gactgaccgt tgtggaaatg tccccctacg gcgttatcgg ggcaattacc 420 ccaagcacaa acccaaccga aaccgtgatt tgtaactcga tcggaatgat cgccgcaggt 480 aacgctgtgg ttttcaacgg ccacccatgc gcaaagaagt gcgttgcatt tgccgtggag 540 atgatcaaca aggcaattat ctcatgcggt ggtcctgaaa acctcgtcac taccattaag 600 aatccaacta tggagtcgct tgatgctatc attaagcacc catcgatcaa gcttctctgt 660 ggaactggcg gccctggcat ggtcaaaacg ctcctgaaca gcgggaaaaa ggcgattgga 720 gccggtgcag gtaatccgcc cgtcatcgtg gacgatacgg cagatattga gaaggccggt 780 cgttccatca tcgaaggctg ctcatttgat aacaacctgc cgtgcattgc tgagaaagaa 840 gtttgcgttt tcgagaacgt tgccgatgac cttatttcca atatgttgaa gaataatgca 900 gtgatcatca acgaagacca agtttccaaa ctgatcgatc tcgtccttca gaaaaacaac 960 gagactcagg aatatttcat taacaagaag tgggtgggca aagacgcaaa gctgttcttg 1020 gatgagattg acgtggagag cccttccaac gtcaagtgca ttatctgtga agtcaacgct 1080 aaccatcctt tcgtgatgac ggaattgatg atgccaatcc tgccgattgt tcgagtaaaa 1140 gacattgacg aagctatcaa gtacgcgaaa atcgccgaac agaaccgcaa gcactctgct 1200 tatatctact ctaagaacat tgacaatctg aaccggtttg aacgggagat cgacactacc 1260 atctttgtca aaaacgcgaa atccttcgct ggcgtgggct atgaagctga gggattcacc 1320 accttcacca ttgcggggag caccggtgaa ggcatcactt ctgcccgcaa cttcacccgc 1380 cagcgccgtt gcgtactcgc cggttaa 1407 <210> 21 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> ldhA KO primer_up <400> 21 atgaaactcg ccgtttatag cacaaaacag tacgacaaga agtacctgca taggtgacac 60 tatagaacgc g 71 <210> 22 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> ldhA KO primer_do <400> 22 ttaaaccagt tcgttcgggc aggtttcgcc tttttccaga ttgcttaagt tagtggatct 60 gatgggtacc 70 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_up <400> 23 tacactaagc atagttgttg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_do <400> 24 ctttcttcat tgtggttctc 20 <210> 25 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> adhE KO primer <400> 25 atggctgtta ctaatgtcgc tgaacttaac gcactcgtag agcgtgtaaa taggtgacac 60 tatagaacgc g 71 <210> 26 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> adhE KO primer <400> 26 ttaagcggat tttttcgctt ttttctcagc tttagccgga gcggcttctt tagtggatct 60 gatgggtacc 70 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer-up <400> 27 caccgcactg actatactct 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer-do <400> 28 gatgaaggct aatgctgtcg 20 <210> 29 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> mdh KO primer_up <400> 29 atgaaagtcg cagtcctcgg cgctgctggc ggtattggcc aggcgcttgc taggtgacac 60 tatagaacgc g 71 <210> 30 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> mdh KO primer_do <400> 30 ttacttatta acgaactctt cgcccagggc gatatctttc ttcagcgtat tagtggatct 60 gatgggtacc 70 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_up <400> 31 ggttcctgat tacggcaatt 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_do <400> 32 attcaggaat atccggcaac 20 <210> 33 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 gctagaattc atgaacttac atgaatatca gg 32 <210> 34 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 gcaaggtacc ttatttcaga acagttttca g 31 <210> 35 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 gaaaactgtt ctgaaataag tcacacagga aacagaattc atggaaataa aagagatg 58 <210> 36 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 ttcagtttga acagttgcat gaattctgtt tcctgtgtga ttagagttcc cagatctc 58 <210> 37 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 aagagatctg ggaactctaa tcacacagga aacagaattc atgcaactgt tcaaactg 58 <210> 38 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 cgactctaga ggatccccgg ttagtacagt cgtctgtaga taccttc 47

Claims (23)

피루베이트로부터 락테이트로 전환하는 활성, 아세틸 CoA를 에탄올로 전환하는 활성, 옥살로아세테이트를 말레이트로 전환하는 활성, 또는 그 조합이 감소되어 있고, 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성이 증가되어 있고, 4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성이 증가되어 있는 것인 미생물.The activity of converting pyruvate to lactate, the activity of converting acetyl CoA to ethanol, the activity of converting oxaloacetate to maleate, or a combination thereof is reduced, and the succinate is reacted with 4-hydroxybutyrate (4HB) Wherein the activity of converting is increased and the activity of converting 4-hydroxybutyrate (4HB) to 1,4-butanediol (1,4-BDO) is increased. 청구항 1에 있어서, 에세리키아 (Escherichia) 속, 코리네박테리움 속, 또는 루멘 박테리아에 속하는 미생물인 것인 미생물.The microorganism according to claim 1, which is a microorganism belonging to the genus Escherichia, genus Corynebacterium, or lumen bacteria. 청구항 1에 있어서, 대장균 (E.coli)인 것인 미생물.The microorganism according to claim 1, which is E. coli. 청구항 1에 있어서, 피루베이트로부터 락테이트로 전환하는 활성은 피루베이트로부터 락테이트로 전환하는 폴리펩티드, 아세틸 CoA를 에탄올로 전환하는 활성은 아세틸 CoA를 에탄올로 전환하는 폴리펩티드, 옥살로아세테이트를 말레이트로 전환하는 활성은 옥살로아세테이트를 말레이트로 전환하는 폴리펩티드, 또는 그 조합의 발현의 감소에 의하여 감소된 것인 미생물.The method of claim 1, wherein the activity of converting pyruvate to lactate is a polypeptide that converts pyruvate to lactate, the activity of converting acetyl CoA to ethanol is a polypeptide that converts acetyl CoA to ethanol, oxaloacetate Wherein the converting activity is reduced by a decrease in the expression of a polypeptide that converts oxaloacetate to a maleate, or a combination thereof. 청구항 4에 있어서, 피루베이트로부터 락테이트로 전환하는 폴리펩티드를 코딩하는 유전자, 아세틸 CoA를 에탄올로 전환하는 폴리펩티드를 코딩하는 유전자, 옥살로아세테이트를 말레이트로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 불활성화되거나 감쇄된 것인 미생물.5. The method according to claim 4, wherein the gene encoding a polypeptide that converts pyruvate to lactate, the gene that encodes a polypeptide that converts acetyl CoA to ethanol, the gene that encodes a polypeptide that converts oxaloacetate to maleate, Wherein the microorganism is inactivated or attenuated. 청구항 1에 있어서, 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드, 숙시닐 CoA를 숙시닉 세미알데히드 (SSA)로 전환하는 폴리펩티드, 숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드, 또는 그 조합의 발현의 증가에 의하여 증가된 것인 미생물. The method of claim 1, wherein the activity of converting the succinate to 4-hydroxybutyrate (4HB) is selected from the group consisting of a polypeptide that converts succinate to succinyl CoA, a polypeptide that converts succinyl CoA to succinic semialdehyde (SSA) (SSA) to 4-hydroxybutyrate (4HB), or a combination thereof. 청구항 1에 있어서, 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닐 CoA를 숙시닉 세미알데히드 (SSA)로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입된 것인 미생물. The method of claim 1, wherein the activity of converting succinate to 4-hydroxybutyrate (4HB) is selected from the group consisting of a gene encoding a polypeptide that converts a succinate to a succinylCoA, a gene that encodes a succinyl-CoA to a succinic semialdehyde (SSA) A gene encoding a polypeptide, a gene encoding a polypeptide that converts succinic semialdehyde (SSA) to 4-hydroxybutyrate (4HB), or a combination thereof. 청구항 1에 있어서, 4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성은 4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드, 4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드, 또는 그 조합의 발현의 증가에 의하여 증가된 것인 미생물. The method according to claim 1, wherein the activity of converting 4-hydroxybutyrate (4HB) to 1,4-butanediol (1,4-BDO) is obtained by reacting 4-hydroxybutyrate (4HB) with 4-hydroxybutyryl- (4HB-CoA), a polypeptide that converts 4-hydroxybutyryl-CoA (4HB-CoA) to 1,4-butanediol (1,4-BDO), or a combination thereof. Microorganisms that are. 청구항 1에 있어서, 4-히드록시부티레이트를 1,4-부탄디올로 전환하는 활성은 4-히드록시부티레이트를 4-히드록시부티릴-CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 4-히드록시부티릴-CoA를 1,4-부탄디올로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입에 의하여 증가된 것인 미생물. The method according to claim 1, wherein the activity of converting 4-hydroxybutyrate to 1,4-butanediol is a gene encoding a polypeptide that converts 4-hydroxybutyrate to 4-hydroxybutyryl-CoA, 4-hydroxybutyryl Wherein the gene encoding a polypeptide that converts -CoA to 1,4-butanediol, or a combination thereof, is increased by introduction. 청구항 1에 있어서, 숙시네이트를 4-히드록시부티레이트로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닐 CoA를 숙시닉 세미알데히드로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닉 세미알데히드를 4-히드록시부티레이트로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입되어 있고, 4-히드록시부티레이트를 4-히드록시부티릴-CoA로 전환하는 활성은 4-히드록시부티레이트를 4-히드록시부티릴-CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 4-히드록시부티릴-CoA를 1,4-부탄디올로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입되어 있고, 피루베이트로부터 락테이트로 전환하는 폴리펩티드를 코딩하는 유전자, 아세틸 CoA를 에탄올로 전환하는 폴리펩티드를 코딩하는 유전자, 옥살로아세테이트를 말레이트로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 불활성화되거나 감쇄된 것인 대장균인 미생물.The method of claim 1, wherein the activity of converting the succinate to 4-hydroxybutyrate is selected from the group consisting of a gene encoding a polypeptide that converts succinate to succinyl CoA, a gene that encodes a polypeptide that converts succinyl CoA to succinic semialdehyde, A gene encoding a polypeptide that converts succinic semialdehyde to 4-hydroxybutyrate, or a combination thereof, is introduced, and the activity of converting 4-hydroxybutyrate to 4-hydroxybutyryl-CoA is 4-hydroxy A gene encoding a polypeptide that converts butyrate to 4-hydroxybutyryl-CoA, a gene that encodes a polypeptide that converts 4-hydroxybutyryl-CoA to 1,4-butanediol, or a combination thereof, A gene encoding a polypeptide that converts pyruvate to lactate, a gene that encodes a polypeptide that converts acetyl CoA to ethanol, Wherein the gene encoding a polypeptide that converts oxaloacetate to maleate, or a combination thereof, is inactivated or attenuated. 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성이 증가되어 있고, 4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성이 증가되어 있는 것인 미생물을 숙시네이트의 존재하에서 배양하는 단계; 및
상기 배양물로부터 1,4-BDO를 회수하는 단계;를 포함하는, 1,4-BDO를 생산하는 방법.The activity of converting succinate to 4-hydroxybutyrate (4HB) is increased and the activity of converting 4-hydroxybutyrate (4HB) to 1,4-butanediol (1,4-BDO) is increased Culturing the microorganism in the presence of succinate; And
Recovering 1,4-BDO from said culture. &Lt; RTI ID = 0.0 &gt; 14. &lt; / RTI &gt; 청구항 11에 있어서, 상기 숙시네이트는 배양 중 피딩되는 것인 방법.12. The method of claim 11, wherein the succinate is fed during culture. 청구항 12에 있어서, 상기 배양은 포화 용존 산소 농도의 약 1 내지 약 100%의 농도에서 수행되는 것인 방법.The method according to claim 12, wherein the culture is performed at a concentration of about 1 to about 100% of the saturated dissolved oxygen concentration. 청구항 11에 있어서, 상기 미생물은 에세리키아 (Escherichia) 속, 코리네박테리움 속, 또는 루멘 박테리아에 속하는 것인 방법.12. The method of claim 11, wherein the microorganism belongs to the genus Escherichia, genus Corynebacterium, or lumen bacteria. 청구항 11에 있어서, 상기 미생물은 대장균 (E.coli)인 것인 방법.12. The method of claim 11, wherein the microorganism is E. coli. 청구항 11에 있어서, 상기 미생물은 피루베이트로부터 락테이트로 전환하는 활성, 아세틸 CoA를 에탄올로 전환하는 활성, 옥살로아세테이트를 말레이트로 전환하는 활성, 또는 그 조합이 감소되어 있는 것인 방법. 12. The method of claim 11, wherein said microorganism is reduced in activity of converting pyruvate to lactate, of converting acetyl CoA to ethanol, of converting oxaloacetate to maleate, or a combination thereof. 청구항 16에 있어서, 상기 미생물은 피루베이트로부터 락테이트로 전환하는 활성은 피루베이트로부터 락테이트로 전환하는 폴리펩티드, 아세틸 CoA를 에탄올로 전환하는 활성은 아세틸 CoA를 에탄올로 전환하는 폴리펩티드, 옥살로아세테이트를 말레이트로 전환하는 활성은 옥살로아세테이트를 말레이트로 전환하는 폴리펩티드, 또는 그 조합의 발현의 감소에 의하여 감소된 것인 방법.[Claim 16] The method according to claim 16, wherein the microorganism is a polypeptide converting pyruvate to lactate, a polypeptide converting pyruvate to lactate, an activity of converting acetyl CoA to ethanol, a polypeptide converting acetyl CoA to ethanol, oxaloacetate Wherein the activity of converting to maleate is reduced by a decrease in the expression of a polypeptide that converts oxaloacetate to maleate, or a combination thereof. 청구항 17에 있어서, 상기 미생물은 피루베이트로부터 락테이트로 전환하는 폴리펩티드를 코딩하는 유전자, 아세틸 CoA를 에탄올로 전환하는 폴리펩티드를 코딩하는 유전자, 옥살로아세테이트를 말레이트로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 불활성화되거나 감쇄된 것인 방법.[19] The method of claim 17, wherein the microorganism is selected from the group consisting of a gene encoding a polypeptide converting pyruvate to lactate, a gene encoding a polypeptide converting acetyl CoA into ethanol, a gene encoding a polypeptide converting oxaloacetate into maleate, Or a combination thereof is inactivated or attenuated. 청구항 11에 있어서, 상기 미생물은 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드, 숙시닐 CoA를 숙시닉 세미알데히드 (SSA)로 전환하는 폴리펩티드, 숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드, 또는 그 조합의 발현의 증가에 의하여 증가된 것인 방법. 12. The method of claim 11, wherein the microorganism is selected from the group consisting of a polypeptide that converts succinate to 4-hydroxybutyrate (4HB), a succinate convert to succinyl CoA, a polypeptide that converts succinyl CoA to succinic semialdehyde (SSA) , A polypeptide that converts succinic semialdehyde (SSA) to 4-hydroxybutyrate (4HB), or a combination thereof. 청구항 11에 있어서, 상기 미생물은 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닐 CoA를 숙시닉 세미알데히드 (SSA)로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입된 것인 방법. 12. The method of claim 11, wherein said microorganism is selected from the group consisting of a gene encoding a polypeptide that converts succinate to succinyl CoA, succinyl-CoA to succinic semialdehyde (SSA) , A gene encoding a polypeptide that converts succinic semialdehyde (SSA) to 4-hydroxybutyrate (4HB), or a combination thereof. 청구항 11에 있어서, 상기 미생물은 4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성은 4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드, 4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드, 또는 그 조합의 발현의 증가에 의하여 증가된 것인 방법. 12. The method according to claim 11, wherein the microorganism is selected from the group consisting of 4-hydroxybutyrate (4HB), 4-hydroxybutyryl (4HB) (4HB-CoA) to the 1,4-butanediol (1,4-BDO), or a combination thereof, which converts the 4-hydroxybutyryl-CoA The way that is increased. 청구항 11에 있어서, 상기 미생물은 4-히드록시부티레이트 (4HB)를 1,4-부탄디올 (1,4-BDO)로 전환하는 활성은 4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드를 코딩하는 유전자, 4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입에 의하여 증가된 것인 방법. 12. The method according to claim 11, wherein the microorganism is selected from the group consisting of 4-hydroxybutyrate (4HB), 4-hydroxybutyryl (4HB) (4HB-CoA) to 1,4-butanediol (1,4-BDO), a gene encoding a polypeptide that converts a 4-hydroxybutyryl-CoA Or a combination thereof is increased by introduction. 청구항 11에 있어서, 상기 미생물은 숙시네이트를 4-히드록시부티레이트 (4HB)로 전환하는 활성은 숙시네이트를 숙시닐 CoA로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닐 CoA를 숙시닉 세미알데히드 (SSA)로 전환하는 폴리펩티드를 코딩하는 유전자, 숙시닉 세미알데히드 (SSA)를 4-히드록시부티레이트 (4HB)로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입되어 있고, 4-히드록시부티레이트를 1,4-부탄디올로 전환하는 활성은 4-히드록시부티레이트 (4HB)를 4-히드록시부티릴-CoA (4HB-CoA)로 전환하는 폴리펩티드를 코딩하는 유전자, 4-히드록시부티릴-CoA (4HB-CoA)를 1,4-부탄디올 (1,4-BDO)로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 도입되어 있고, 피루베이트로부터 락테이트로 전환하는 폴리펩티드를 코딩하는 유전자, 아세틸 CoA를 에탄올로 전환하는 폴리펩티드를 코딩하는 유전자, 옥살로아세테이트를 말레이트로 전환하는 폴리펩티드를 코딩하는 유전자, 또는 그 조합이 불활성화되거나 감쇄된 것인 대장균인 방법.


12. The method of claim 11, wherein said microorganism is selected from the group consisting of a gene encoding a polypeptide that converts succinate to succinyl CoA, succinyl-CoA to succinic semialdehyde (SSA) , A gene encoding a polypeptide that converts succinic semialdehyde (SSA) to 4-hydroxybutyrate (4HB), or a combination thereof is introduced, and 4-hydroxybutyrate is substituted with 1, The activity of converting 4-butanediol into a 4-hydroxybutyryl-CoA (4HB-CoA) gene encoding a polypeptide that converts 4-hydroxybutyrate (4HB) to 4-hydroxybutyryl- CoA) into 1,4-butanediol (1,4-BDO), or a combination thereof, and a gene encoding a polypeptide that converts pyruvate to lactate, a gene coding for a polypeptide encoding acetyl CoA Wherein the gene encoding a polypeptide that converts to oxaloacetate to ethanol is a gene that encodes a polypeptide that converts oxaloacetate to maleate, or a combination thereof is inactivated or attenuated.


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